help button home button Endocrine Society Molecular Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2006-0178
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
20/12/3400    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Syme, C. A.
Right arrow Articles by Bisello, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Syme, C. A.
Right arrow Articles by Bisello, A.
Molecular Endocrinology 20 (12): 3400-3411
Copyright © 2006 by The Endocrine Society

Caveolin-1 Regulates Cellular Trafficking and Function of the Glucagon-Like Peptide 1 Receptor

Colin A. Syme, Lei Zhang and Alessandro Bisello

Division of Endocrinology and Metabolism, Departments of Medicine (C.A.S., L.Z., A.B.) and of Pharmacology (A.B.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Address all correspondence and requests for reprints to: Alessandro Bisello, Division of Endocrinology and Metabolism, University of Pittsburgh School of Medicine, E1140 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, Pennsylvania 15261. E-mail: BiselloA{at}dom.pitt.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The glucagon-like peptide 1 receptor (GLP-1R) mediates important effects on ß-cell function and glucose homeostasis and is one of the most promising therapeutic targets for type 2, and possibly type 1, diabetes. Yet, little is known regarding the molecular and cellular mechanisms that regulate its function. Therefore, we examined the cellular trafficking of the GLP-1R and the relation between receptor localization and signaling activity. In resting human embryonic kidney 293 and insulinoma MIN6 cells, a fully functional green fluorescent protein-tagged GLP-1R was localized both at the cell membrane and in highly mobile intracellular compartments. Real-time confocal fluorescence microscopy allowed direct visualization of constitutive cycling of the receptor. Overexpression of K44A-dynamin increased the number of functional receptors at the cell membrane. Immunoprecipitation, sucrose sedimentation, and microscopy observations demonstrated that the GLP-1R localizes in lipid rafts and interacts with caveolin-1. This interaction is necessary for membrane localization of the GLP-1R, because overexpression of a dominant-negative form of caveolin-1 (P132L-cav1) or specific mutations within the putative GLP-1R’s caveolin-1 binding domain completely inhibited GLP-1 binding and activity. Upon agonist stimulation, the GLP-1R underwent rapid and extensive endocytosis independently from arrestins but in association with caveolin-1. Finally, GLP-1R-stimulated activation of ERK1/2, which involves transactivation of epidermal growth factor receptors, required lipid raft integrity. In summary, the interaction of the GLP-1R with caveolin-1 regulates subcellular localization, trafficking, and signaling activity. This study provides further evidence of the key role of accessory proteins in specifying the cellular behavior of G protein-coupled receptors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GLUCAGON-LIKE PEPTIDE 1 (GLP-1) acts upon the glucagon-like peptide 1 receptor (GLP-1R) to potentiate glucose-dependent insulin secretion by pancreatic ß-cells (1, 2, 3). GLP-1 also induces ß-cell proliferation and is protective against apoptosis in both ß-cells and neurons (4, 5). Exendin-4, an exogenous GLP-1R agonist isolated from the Gila monster lizard has very similar effects (3) and has recently been approved for the treatment of type 2 diabetes.

The GLP-1R belongs to the class B family of G protein-coupled receptors (GPCRs), which includes receptors for several medium-sized peptide hormones, such as PTH, secretin, glucagon, and calcitonin. Class B GPCRs are characterized by a common topology of the ligand-receptor complex, and by their ability to couple multiple G proteins. The downstream signaling pathways have been investigated extensively for the GLP-1R (6). Previous studies have demonstrated that agonist stimulation of the GLP-1R induces cAMP and Ca2+ signaling (7, 8). Also, GLP-1 and exendin-4 stimulate activation of phosphatidylinositol 3-kinase (PI3K) and ERK1/2 (9, 10, 11, 12). Surprisingly, very little is known about the regulation of membrane expression and trafficking of the GLP-1R. Thorens and co-workers (13, 14) demonstrated by using radioiodinated GLP-1 that GLP-1R internalization occurs and is dependent on phosphorylation of several residues within the C-terminal tail. Moreover, in contrast to most class B GPCRs, GLP-1R resensitization occurs rapidly (15), suggesting that trafficking events may significantly regulate GLP-1R function. Despite the evidence in cell systems, it is uncertain whether the GLP-1R desensitizes in vivo. A 48-h infusion of GLP-1 in rats resulted in increased insulin secretion and ß-cell proliferation (16), indicating that prolonged stimulation of the GLP-1R induces appropriate biological responses. Moreover, transgenic mice overexpressing exendin-4 showed significantly increased insulin levels after oral glucose administration (17), indicating that incretin responses were not suppressed by the continuous presence of exendin-4. However, no evidence of increased ß-cell mass was observed in mice overexpressing exendin-4 (17). There is therefore an apparent discordance between studies in cell systems and in animal models. At present, the molecular mechanisms regulating GLP-1R function remain unclear.

After agonist stimulation, the majority of GPCRs traffic to clathrin-coated pits, in a ß-arrestin-dependent fashion and are internalized by endocytosis (18). However, some GPCRs preferentially localize to and/or internalize via specialized lipid raft/caveolae microdomains of the plasma membrane. These include, among others, the angiotensin II type 1 (AT1), endothelin A (ETA), and somatostatin receptors (19). Lipid raft-mediated endocytosis can lead to the pinching off of caveolae and their fusion with large intracellular organelles called caveosomes (20). It is clear that several factors control the endocytic pathway chosen by a specific GPCR. For instance, whereas the endothelin ETA receptor resides in lipid rafts and is internalized via caveolae (21), the ß2-adrenergic receptor leaves the lipid rafts after agonist binding to be internalized via clathrin-coated pits (22, 23). Furthermore, disruption of lipid rafts by cholesterol depletion can switch caveolae-mediated endocytosis to a clathrin-coated pit internalization pathway, as in the case of the endothelin receptor ETA (24).

A feature of these receptors is their ability to bind caveolin-1. Caveolin-1, a 21- to 24-kDa protein, is the principal protein component of caveolae. It can interact with a number of signaling molecules including G proteins, receptor tyrosine kinases, and GPCRs via a common caveolin-binding motif ({Phi}X{Phi}XXXX{Phi} and {Phi}XXXX{Phi}XX{Phi}, where {Phi} is an aromatic amino acid) (25, 26).

In this study we used both human embryonic kidney (HEK)293 cells (which do not express GLP-1R and therefore allow the characterization of mutated forms of the receptor) and MIN6 cells, a mouse insulinoma cell line that expresses endogenous GLP-1R (27), to investigate the mechanisms regulating the cellular localization and trafficking of the GLP-1R. We demonstrate that GLP-1R interacts with caveolin-1 in an association that is necessary for receptor trafficking to the cell membrane. Furthermore, we show that the subcellular localization of the GLP-1R is important for the stimulation of ERK1/2 signaling via transactivation of epidermal growth factor (EGF) receptor.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Characterization of the GLP-1R-GFP
Expression and signaling of wild-type and C-terminally GFP epitope-tagged GLP-1R were characterized in HEK293 cells. Membrane expression and affinity of the GLP-1R and GLP-1R-GFP expressed in HEK293 cells were determined by radioreceptor binding assays using HPLC-purified [125I]GLP-1 (Fig. 1AGo). In these cells, expressing approximately 50,000 receptors per cell, the IC50 was 3.7 ± 0.1 nM and 7.5 ± 1.5 nM for wild-type and GFP-tagged GLP-1R, respectively. Agonist stimulation of both wild-type and GFP-tagged GLP-1R resulted in a dose-dependent increase in cAMP with an EC50 of approximately 1 nM and identical maximal increases in cAMP (Fig. 1BGo). Next, regulation of cAMP activity in response to agonist stimulation using HEK293 cells stably expressing GLP-1R-GFP was determined (Fig. 1CGo). Exposure to GLP-1 (10 nM) for 30 min resulted in a 28-fold increase in acute levels of cAMP accumulation over basal. After a 2-h washout period the residual cAMP accumulation was 4.9 ± 0.1% of the acute response in the presence of GLP-1 (P < 0.0001), indicating that termination of the original signal occurred. In similarly treated cells, full cAMP activity (99 ± 1% of the acute response) was measured after rechallenge with 10 nM GLP-1, indicating that the receptor fully resensitized. Finally, in HEK293 cells stably expressing the GLP-1R, stimulation with 100 nM GLP-1 induced rapid and transient increases in intracellular calcium (Fig. 1DGo).


Figure 1
View larger version (21K):
[in this window]
[in a new window]
  		Fig. 1. Characterization of the GLP-1R in HEK293 Cells

A, Binding. Radioreceptor binding assays were performed in HEK293 transiently transfected with either wild-type ({circ}) or GFP-tagged GLP-1R ({square}) (0.4 µg DNA/well) using radioiodinated GLP-1 and competition with GLP-1. Each point represents the mean ± SEM from triplicate experiments. B, cAMP accumulation. HEK293 cells were transiently transfected with either wild-type ({circ}) or GFP-tagged GLP-1R ({square}) (0.4 µg DNA/well) and stimulated with the indicated concentrations of exendin-4 for 30 min. Each point represents the mean ± SEM from triplicate experiments. C, Regulation of cAMP signaling. HEK-293 stably expressing GLP-1R were treated with either vehicle (0.1% BSA) or GLP-1 (10 nM) for 30 min, followed by a 2-h washout at 37 C. Rechallenged and residual cAMP accumulation was then measured for 30 min in the presence of IBMX (1 mM) with or without reexposure to GLP-1, respectively. Results are presented as mean ± SEM from three independent experiments. D, Intracellular calcium. Calcium signaling was measured in HEK293 cells stably expressing GLP-1R. Cells were loaded with the calcium-sensitive dye Fura2-AM, and the increases in intracellular calcium concentration in response to 100 nM GLP-1 and thrombin were monitored spectroscopically. AU, Arbitrary units.

 
Collectively, these data confirm that the GLP-1R (both wild-type and GFP-tagged) is properly expressed in HEK293 cells, and its signaling is consistent with that determined in other cell systems (1, 2, 7, 8).

Cellular Localization of the GLP-1R
The subcellular localization of the GLP-1R-GFP was determined in HEK293 and MIN6 cells by real-time confocal fluorescence microscopy. In resting cells, the GLP-1R-GFP localized at both the plasma membrane and in mobile cytosolic compartments. Live-cell confocal imaging of cells expressing the GLP-1R-GFP allowed direct visualization of the receptor movement to and from the cell membrane. Figure 2Go shows series of micrographs of a portion of the plasma membrane of an HEK293 cell (upper panels) and a MIN6 cell (lower panels) maintained in media at 37 C. The appearance and disappearance of clusters of GLP-1R-GFP at the cell membrane (indicated by the arrows) can be appreciated. To determine the identity of the cytosolic structures, we performed cell fractionation experiments. Triton-soluble and insoluble fractions, the latter representing lipid rafts and caveolae, were obtained from HEK293 cells expressing the GLP-1R-GFP, resolved on SDS-PAGE and immunoblotted for GFP. As shown in Fig. 3AGo, the GLP-1R-GFP was specifically detected in the Triton-insoluble fraction. Furthermore, to determine whether GLP-1R is present in caveolae we used a well-established fractionation method based on the specific buoyancy of caveolin-rich membranes in sucrose density gradients (28, 29). As shown in Fig. 3BGo, caveolin-1 is predominantly localized to fractions 4 and 5, and there is significant overlap with GLP-1R-GFP localization. Collectively, these results suggest that the GLP-1R localizes in membrane lipid rafts and caveosomes.


Figure 2
View larger version (32K):
[in this window]
[in a new window]
  		Fig. 2. Cellular Localization of the GLP-1R

Living HEK293 (top panels) or MIN6 (lower panels) cells expressing GLP-1R-GFP were analyzed by confocal fluorescence microscopy at 37 C. Micrographs were collected every 4 sec. The GLP-1R-GFP localizes at the cell membrane and in mobile intracellular structures. The appearance and disappearance of GLP-1R-GFP clusters at the cell membrane are indicated by the arrows. s, Seconds.

 

Figure 3
View larger version (56K):
[in this window]
[in a new window]
  		Fig. 3. The GLP-1R Localizes to Lipid Rafts/Caveolae under Basal Conditions

A, Triton X-100 soluble and insoluble fractions were obtained from HEK293 cells transiently transfected with either pcDNA3 or the GLP-1R-GFP as described in Materials and Methods. Equal amounts of proteins from the soluble and insoluble fractions (Sol and Ins, respectively) were analyzed by immunoblotting for GFP. B, HEK293 cells stably transfected with GLP-1R-GFP were homogenized in buffer containing 1% Triton X-100 as described in Materials and Methods. After separation by centrifugation in a sucrose gradient, fractions were collected and equal volumes were analyzed by Western blot with specific antibody against GFP (upper panel) or caveolin-1 (lower panel).

 
The GLP-1R Interacts with Caveolin-1
Because the previously described experiments suggested that the GLP-1R localizes in caveolae, we examined its possible interaction with caveolin-1. Examination of the GLP-1R sequence revealed a classical caveolin-1 binding motif within the second intracellular loop (247EGVYLYTLLAFSVF260).

The interaction between GLP-1R-GFP and caveolin-1 was determined by immunoprecipitation (Fig. 4Go). The GLP-1R-GFP and caveolin-1 were transiently transfected in HEK293 cells. Cell lysates were immunoprecipitated with monoclonal GFP antibody conjugated to agarose, followed by immunoblotting for caveolin-1. In these conditions, caveolin-1 was specifically immunoprecipitated (Fig. 4AGo; lower panel, lane 2). Two mutations within the putative caveolin-1 binding domain of the GLP-1R-GFP (E247A and Y250/252A) were generated. The mutated receptors were properly synthesized and, in the case of E247A-GFP, expressed at levels comparable to the wild-type GLP-1R-GFP (Fig. 4Go, A and B; upper panels). Introduction of these mutations significantly reduced the amount of immunoprecipitated caveolin-1 (for E247A-GFP the ratio receptor/caveolin-1 was 53 ± 13% of wild type; P < 0.03, n = 3) (lower panel, lanes 3 and 4). Moreover, to determine whether the interaction between GLP-1R and caveolin-1 occurs at endogenous caveolin-1 levels, we performed the reverse experiment from cells expressing GLP-1R-GFP, E247A-GFP, and Y250/252A-GFP by immunoprecipitating endogenous caveolin-1. Again, specific coimmunoprecipitation of GLP-1R-GFP and caveolin-1 could be demonstrated for the GLP-1R-GFP, whereas little, if any, of the two mutated receptors coimmunoprecipitated with caveolin-1 (Fig. 4BGo, lower panel).


Figure 4
View larger version (49K):
[in this window]
[in a new window]
  		Fig. 4. The GLP-1R Interacts with Caveolin-1

A, HEK293 cells expressing either the wild-type GLP-1R-GFP, the E247A-GFP, or the Y250/252A-GFP receptors and caveolin-1, as indicated, were lysed, and immunoprecipitation experiments were performed with anti-GFP antibody followed by SDS-PAGE on a 12% gel and immunoblotting with anticaveolin-1 antibody (lower panel). B, HEK293 cells expressing either the wild-type GLP-1R-GFP, the E247A-GFP, or the Y250/252A-GFP receptors were lysed, and immunoprecipitation experiments were performed with anticaveolin-1 antibody followed by SDS-PAGE on a 7.5% gel and immunoblotting with anti-GFP antibody (lower panel). In both cases, the amount of GFP-tagged receptors in each sample was verified by immunoblotting with anti-GFP antibody (upper panel). WT, Wild type; IP, immunoprecipitation; IB, immunoblot.

 
The Interaction between GLP-1R and Caveolin-1 Is Necessary for Receptor Trafficking to the Cell Membrane
The presence of a significant population of GLP-1R in mobile intracellular compartments suggests that the interaction with caveolin-1 may be functionally important for the trafficking of the receptor to the cell membrane. To examine this possibility, we took advantage of the availability of a dominant-negative form of caveolin-1 (P132L-cav1). P132L-cav1 sequesters endogenous caveolins in the Golgi, thereby effectively inhibiting caveolae formation (30). Coexpression of wild-type GLP-1R with P132L-cav1 in HEK293 cells completely abolished [125I]GLP-1 binding (Fig. 5AGo), indicating that caveolin-1 function is necessary for the correct localization of the GLP-1R to the cell membrane. To further support this observation, membrane expression of the wild-type GLP-1R and of the E247A and Y250/252A mutants was determined by radioreceptor binding assays (Fig. 5BGo). As described above, [125I]GLP-1 specifically bound the wild-type GLP-1R-GFP. However, virtually no specific [125I]GLP-1 binding to either E247A or Y250A/Y252A was detected (2.9 ± 0.7% and 4.5 ± 2.0% of GLP-1R binding, respectively). As expected, whereas stimulation of wild-type GLP-1R with GLP-1 (100 nM) induced robust cAMP accumulation (23-fold over basal), GLP-1 failed to induce increases in cAMP in cells expressing either E247A or Y250A/Y252A. In contrast, mutations introduced in residues flanking the tyrosines of the caveolin-1 binding motif (V249A or T253A) did not affect cAMP signaling of GLP-1R-GFP in response to agonist (Fig. 5CGo), confirming that the impaired expression of the E247A and Y250/252A mutants is not caused by a general, nonspecific misfolding of the receptor. Identical results were obtained in HEK293 cells stably expressing GLP-1R, E247A, and Y250/252A receptors with or without GFP (data not shown). Furthermore, fluorescence microscopy analysis showed that whereas the wild-type GLP-1R-GFP was localized both at the cell membrane and intracellularly and colocalized with caveolin-1, the E247A mutant was found exclusively intracellularly and did not significantly colocalize with caveolin-1 (Fig. 5DGo). Collectively, these data show that the interaction between GLP-1R and caveolin-1 is necessary for receptor trafficking and localization to the cell membrane.


Figure 5
View larger version (14K):
[in this window]
[in a new window]
  		Fig. 5. The Interaction between GLP-1R and Caveolin-1 Is Necessary for Receptor Trafficking to the Cell Membrane

A, The GLP-1R (0.2 µg DNA/well) was coexpressed with either pcDNA3 or the dominant-negative caveolin-1 P132L-cav1 (0.5 µg DNA/well). Membrane expression was measured by [125I]GLP-1 binding. No binding was measured in cells expressing P132L-cav1. B, Membrane expression of GLP-1R or the mutants E247A and Y250/252A transiently expressed in HEK293 cells (0.4 µg) was measured by [125I]GLP-1 binding. No binding was measured in cells expressing either E247A or Y250/252A. C, Adenylyl cyclase activity of GLP-1R or the mutants E247A, Y250/252A, V249A, and T253A transiently expressed in HEK293 cells (0.4 µg) in response to vehicle (black bars) or 100 nM exendin-4 (white bars). D, HEK293 cells stably expressing either the wild-type GLP-1R-GFP or the E247A-GFP receptors were fixed, immunostained for caveolin-1 (in red), and examined by confocal fluorescence microscopy. The GLP-1R-GFP is expressed predominantly on the cell membrane where it colocalizes with caveolin-1 (in yellow). In contrast, the mutant E247A-GFP is found only intracellularly and does not colocalize with caveolin-1.

 
Constitutive Cycling of the GLP-1R
The observation of the dynamic behavior of GLP-1R in unstimulated cells (Fig. 2Go) strongly suggests that the GLP-1R undergoes constitutive trafficking. Dynamin is a key molecule involved in the final steps of both endosome and caveolae formation, and its function is necessary for the internalization of membrane molecules via both pathways. Therefore, we hypothesized that expression of a dominant-negative form of dynamin (K44A-dynamin), thus inhibiting endocytosis, will increase the amount of GLP-1R on the cell membrane. As expected, coexpression of GLP-1R and K44A-dynamin resulted in a 2.5-fold increase of the specific GLP-1 binding sites compared with control cells (Fig. 6AGo). Moreover, cAMP generation in response to exendin-4 was significantly higher in cells expressing K44A-dynamin (Fig. 6BGo).


Figure 6
View larger version (11K):
[in this window]
[in a new window]
  		Fig. 6. Constitutive Trafficking of the GLP-1R

GLP-1R (0.2 µg DNA/well) was coexpressed with either pcDNA3 or K44A-dynamin (0.5 µg DNA/well) in HEK293 cells. A, Membrane expression measured by [125I]GLP-1 binding. *, P < 0.0001 vs. pcDNA3 (n = 6), two-tailed Student’s t test. B, cAMP activity in response to exendin-4 in cells transfected with GLP-1R and either pcDNA3 (white bars) or K44A-dynamin (black bars). *, P < 0.002 and #, P < 0.02 vs. pcDNA3 (n = 6); two-tailed Student’s t test.

 
GLP-1R Internalization
Little is known regarding the mechanisms of agonist-induced internalization of the GLP-1R. Therefore, we monitored the trafficking of the GLP-1R-GFP stably expressed in HEK293 cells in response to agonist stimulation (Fig. 7Go). In resting confluent cells the GLP-1R-GFP was mostly localized on the cell membrane and to a lesser extent intracellularly, and it colocalized with caveolin-1 (Fig. 7AGo, top panels). Stimulation with 10 nM exendin-4 induced rapid and extensive internalization of the GLP-1R-GFP. After 15 min, virtually all of the membrane receptor was internalized. The internalized GLP-1R-GFP colocalized with caveolin-1 (Fig. 7AGo, lower panels). Similar observations were made in MIN6 cells transiently expressing the GLP-1R-GFP (Fig. 7BGo). Because many GPCRs internalize in association with ß-arrestins, we investigated whether or not the cellular distribution of GFP-tagged ß-arrestin2 (ß-Arr2-GFP) changed in response to GLP-1R stimulation. In resting HEK293 cells transiently expressing ß-Arr2-GFP and the GLP-1R, the GFP fluorescence was evenly distributed throughout the cytoplasm (Fig. 8Go). Upon receptor activation by either exendin-4 or GLP-1 (up to 100 nM), the cellular distribution of ß-Arr2-GFP did not change. In contrast, parallel experiments carried out in HEK293 cells expressing the PTH 1 receptor (PTH1R) demonstrated the complete relocalization of ß-Arr2-GFP in response to PTHrP, as we previously reported (31, 32) (Fig. 8Go).


Figure 7
View larger version (24K):
[in this window]
[in a new window]
  		Fig. 7. GLP-1R-GFP Internalization

A, HEK293 cells stably expressing GLP-1R-GFP (green) were incubated in the absence (basal) or presence of 10 nM exendin-4 for 15 min, as indicated. Cells were fixed, immunostained for caveolin-1 (in red), and examined by confocal fluorescence microscopy. After agonist stimulation, extensive internalization of the GLP-1R-GFP occurs, and the internalized GLP-1R-GFP colocalizes with caveolin-1. B, MIN-6 cells transiently expressing the GLP-1R-GFP were treated in the absence (basal) or presence of 10 nM exendin-4 for the indicated times at 37 C. Cells were fixed and examined by confocal fluorescence microscopy.

 

Figure 8
View larger version (8K):
[in this window]
[in a new window]
  		Fig. 8. GLP-1R Endocytosis Is Independent from ß-Arrestins

HEK293 cells were cotransfected with ß-Arr2-GFP (0.1 µg) and either GLP-1R or PTH1R (0.4 µg), as indicated. Cells were treated in the absence (basal) or presence of 100 nM exendin-4, GLP-1, or PTHrP(1–36) for 15 min at 37 C, fixed, and examined by confocal fluorescence microscopy. In cells expressing GLP-1R, neither exendin-4 nor GLP-1 induced any relocalization of ß-Arr2-GFP. In contrast, stimulation of cells expressing the PTH1R with PTHrP(1–36) caused ß-Arr2-GFP redistribution to endosomes.

 
Activation of ERK1/2 by the GLP-1R Depends on Lipid Raft Integrity
In MIN6 cells, which express endogenous GLP-1R, stimulation of ERK1/2 activity (maximal stimulation at 2–5 min after stimulation with 10 nM exendin-4) occurred in response to both exendin-4 (Fig. 9AGo) and GLP-1 (data not shown). Previous studies have implicated transactivation of EGF receptors by ß-cellulin as a mechanism leading to PI3K activation by GLP-1 (12). To determine whether a similar mechanism leads to GLP-1R-mediated ERK1/2 activation, we tested the effect of tyrphostin AG1478, a selective EGF receptor inhibitor, on ERK1/2 activation in response to exendin-4. As shown in Fig. 9BGo, GLP-1R-mediated activation of ERK1/2 was partly dependent on EGF receptor transactivation, because pretreatment with AG1478 (100 nM) strongly reduced exendin-4-induced ERK1/2 phosphorylation. In the same experimental conditions, AG1478 fully inhibited the response to EGF (10 ng/ml). Identical results were obtained in HEK293 cells stably expressing the GLP-1R-GFP (data not shown). We then tested whether the localization of the GLP-1R in lipid rafts is important for the stimulation of ERK1/2 signaling (Fig. 9Go). In MIN6 cells, both exendin-4 and EGF stimulated ERK1/2 phosphorylation. As expected, cholesterol depletion with 10 mM isopropyl-ß-cyclodextrin resulted in increases in EGF-stimulated ERK1/2 activity (33, 34). In striking contrast, however, cyclodextrin inhibited GLP-1R-mediated ERK1/2 phosphorylation by 50% (P < 0.05, n = 3) (Fig. 9CGo). This inhibition is particularly remarkable because, as shown previously, the GLP-1R activates ERK1/2 in part via activation of the EGF receptor, which is increased after cholesterol depletion.


Figure 9
View larger version (28K):
[in this window]
[in a new window]
  		Fig. 9. ERK1/2 Activation in MIN6 Cells

A, Time-course of ERK1/2 activation. MIN-6 cells were stimulated with vehicle (0.1% BSA in PBS) or with 10 nM exendin-4 for the indicated times. B, GLP-1R-mediated activation of ERK1/2 is partly dependent on EGF receptor transactivation. MIN-6 cells were treated with or without the EGF receptor inhibitor tyrphostin AG1478 (100 nM) for 15 min, followed by treatment with 10 nM exendin-4 or 10 ng/ml EGF for 5 min, as indicated. C, Effect of cholesterol depletion on activation of ERK1/2. Top panel, MIN6 cells were treated with or without isopropyl-ß-cyclodextrin (10 mM) for 30 min, followed by treatment with 10 nM exendin-4 (Ex-4) or 1 ng/ml EGF for 5 min, as indicated. Lower panel, Data from three independent experiments are expressed as fold-over-basal (untreated cells) of the ratio pERK/ERK. *, P < 0.05 vs. exendin-4 (n = 3); two-tailed Student’s t test. In all cases, phosphorylated and total ERK1/2 were determined by Western blot.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study we describe an investigation of the mechanisms that govern GLP-1R membrane expression and trafficking, and of the relation between the subcellular localization of the GLP-1R and its signaling activity. Our findings show that: 1) in unstimulated cells, the GLP-1R is localized on the cell membrane and in highly mobile intracellular compartments where it associates with caveolin-1; 2) the interaction with caveolin-1 is necessary for the proper targeting of the GLP-1R, because inhibition of this interaction by either dominant-negative caveolin-1 or mutations within a putative caveolin-binding domain of the GLP-1R abrogate membrane expression; 3) the GLP-1R constitutively cycles between cytoplasm and cell membrane; 4) in response to agonist stimulation, the GLP-1R is rapidly internalized in association with caveolin-1; and 5) the localization of the GLP-1R in lipid rafts affects its ability to induce transactivation of the EGF receptor leading to stimulation of ERK1/2 phosphorylation.

A key feature of this study is the characterization of the interaction between the GLP-1R and caveolin-1 and its fundamental role in regulating cellular localization and trafficking of the GLP-1R. The scaffolding domain of caveolin-1 binds to specific motifs rich in aromatic residues (25), and caveolin-binding motifs have been identified in an increasing number of GPCRs, including AT1 (29, 35), and endothelin (36) receptors. The GLP-1R possesses a canonical caveolin-binding sequence at the interface of the third transmembrane and the second intracellular loop. Mutation of two tyrosine residues within this motif to alanine (Y250A/Y252A) results in a receptor that fails to interact with caveolin-1 and is not expressed on the cell surface. Previous studies have shown that similar mutations in the insulin and AT1 receptors reduce membrane expression (29, 35, 37). Interestingly, mutation of E247 to alanine produced identical results, suggesting that residues flanking the caveolin-binding domain may participate in the interaction. Similar to our findings with the E247A and Y250/252A GLP-1R mutants, the AT1 receptor is trapped in intracellular compartments in cells lacking caveolin-1 (35). Although we cannot completely exclude possible effects of the mutations on receptor folding, we note that identical effects were observed after inhibition of caveolin-1 function by the dominant-negative P132L-cav1. Collectively, these observations strongly suggest that caveolin-1 may function as a molecular chaperone involved in GPCR routing to the plasma membrane.

Studies by Widmann et al. (15) suggested a continuous cycling of the GLP-1R to and from the cell membrane. Consistent with these observations, we show that the GLP-1R constitutively internalizes in a dynamin-dependent manner. The functional significance of the constitutive cycling of the GLP-1R is still unclear. Desensitization and resensitization of GLP-1R signaling has been demonstrated in vitro in several cell systems (Fig. 1Go and Refs. 13, 17 and 38, 39, 40, 41). However, whether the GLP-1R desensitizes in vivo is uncertain (16, 17). Our studies suggest that whereas the GLP-1R indeed undergoes desensitization in response to agonists, the intracellular pool of GLP-1R in equilibrium with the membrane population may function as an efficient mechanism for resensitization. Consistent with this hypothesis, the kinetics of recycling of the GLP-1R [t1/2 of 15 min (15)] is remarkably fast when compared with that of other class B GPCRs such as the PTH type 1 and the GLP-2 receptors (31, 42).

A number of GPCRs have been localized in lipid rafts and/or caveolae (19). The localization of GPCRs in lipid rafts and caveolae has specific and distinct functions. In some cases, lipid rafts regulate receptor stability [as for the endothelin ETB (36) and calcium sensing receptors (43, 44)], signaling [as for the muscarinic (45) and adrenergic receptors (22, 23)], and endocytosis [as for GLP-2 (42), ß1-adrenergic (23), and cholecystokinin type A (46) receptors]. From this perspective, it is interesting to note that the localization of the GLP-1R in lipid rafts profoundly affects its ability to stimulate ERK1/2 phosphorylation through transactivation of EGF receptors. This signaling pathway, together with the parallel PI3K pathway, is of paramount importance for the proliferative, and perhaps the antiapoptotic, effects of GLP-1 in ß-cells (12, 47, 48, 49).

We found that upon agonist stimulation the GLP-1R is internalized in association with caveolin-1. Interestingly, and in contrast to many other GPCRs, the GLP-1R does not appear to utilize ß-arrestin2 for endocytosis when expressed in HEK293 cells. At present, we cannot exclude a possible involvement of ß-arrestin-1 and/or differences in the endocytic pathway in different cell types (50), but it is clear that the mechanisms of GPCR endocytosis vary considerably, even among remarkably similar receptors, and it is tempting to speculate that these differences may underlie specific signaling events. Considerable evidence over the past few years has clearly shown that trafficking events, especially endocytosis, are directly related to the activation of signal transduction pathways, often in a G protein-independent manner (18, 51, 52).

In conclusion, our studies show that the GLP-1R is regulated in a complex manner, involving mechanisms that are quite unusual among GPCRs. Given the physiological importance and therapeutic potential of the GLP-1R, understanding the mechanisms associated with its expression, signaling, and regulation may contribute to the development of therapeutic agents directed to the treatment of type 2, and possibly type 1, diabetes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Anti-GFP mouse monoclonal and anticaveolin-1 rabbit polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat antirabbit and antimouse antibodies were obtained from Cell Signaling Technology (Beverly, MA). Both GLP-1(7–36) and exendin-4 were purchased from Bachem (Torrance, CA). Alexa635-tagged goat antirabbit antibody was purchased from Molecular Probes (Eugene, OR). Protease inhibitor cocktail set I was obtained from Calbiochem (La Jolla, CA). DMEM was from Cambrex Bioscience (Walkersville, MD). Fetal bovine serum, penicillin-streptomycin, and geneticin were from Invitrogen (Carlsbad, CA). All restriction enzymes were obtained from New England Biolabs (Beverly, MA). All other reagents were from Sigma-Aldrich (St. Louis, MO).

Molecular Biology
C-terminally GFP-tagged GLP-1R was generated, using the pcDNA3-GLP-1R expression plasmid, by deletion of the stop codon using the QuikChange site-directed mutagenesis strategy developed by Stratagene (La Jolla, CA). GLP-1R was then subcloned in-frame from pcDNA3 into HindIII-ApaI-digested pEGFP-N2 (BD Biosciences, Palo Alto, CA) using restriction sites for HindIII and ApaI flanking the 5'- and 3'-ends of the receptor, respectively. Mutations of the GLP-1R caveolin-binding domain (E247A, V249A, Y250A/Y252A, and T253A) were introduced into the GFP epitope-tagged construct by site-directed mutagenesis. The fidelity of all constructs was confirmed by sequencing (ABI PRISM 377 automated sequencer, University of Pittsburgh) and subsequent sequence alignment (NCBI BLAST) with human GLP-1R (GenBank accession no. NM_002062.2).

Cell Culture
Human embryonic kidney cells (HEK293) were cultured in DMEM (Cambrex BioScience) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and 1% penicillin-streptomycin (Invitrogen) in a humidified 5% CO2-95% air incubator at 37 C. MIN6 cells (27), a mouse insulinoma cell line expressing endogenous GLP-1R, were cultured in DMEM, 10% FBS, 50 µM 2-mercaptoethanol, 0.1 mM HEPES, 1 mM sodium pyruvate, and 1% penicillin-streptomycin. Cells were transfected using Fugene 6 (Roche, Indianapolis, IN), as previously described (31). Stable cell lines expressing wild-type or mutant GLP-1R and GLP-1R-GFP were generated by antibiotic selection (1 mg/ml G418) 48 h after transfection. Selection was typically complete within 14 d.

Radioligand Binding
Radioiodination and HPLC purification of GLP-1(7–36) was carried out using the Iodogen method as previously described (32). Radioligand receptor binding assays were performed as previously described (53, 54). The binding affinity of GLP-1 was determined by Scatchard analysis of radioreceptor binding assays performed in triplicate.

Adenylyl Cyclase Activity and Intracellular Calcium Determinations
cAMP accumulation was determined in subconfluent cell cultures in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) as previously described (31, 54). Cells were pretreated without or with either GLP-1 or exendin-4 for 30 min, followed by a 2-h washout at 37 C in cell culture medium containing [3H]adenine (2 µCi/ml). Subsequently, the acute response and resensitization were determined by either GLP-1 or exendin-4 stimulation for 30 min of unstimulated cells and agonist-pretreated cells, respectively. Residual cAMP accumulation was measured for 30 min in the presence of IBMX (1 mM). For dose-response experiments, cells were treated with exendin-4 acutely (30 min) at increasing concentrations. In all cases, reactions were stopped with 1.2 M trichloroacetic acid, and cAMP was isolated by the two-column chromatographic method (55). The stimulation of increases in intracellular calcium levels by GLP-1 was assessed spectroscopically in Fura-2-loaded HEK293 cells stably transfected with GLP-1R as described (31, 54).

Fluorescence Microscopy
For fluorescence microscopy studies, cells were plated on 25-mm glass coverslips (1.5 x 105 cells per coverslip), transfected with the indicated GFP-tagged GLP-1R or ß-Arr2-GFP, and cultured for 24 h. For live-cell microscopy, cells grown on glass coverslips were mounted in an open-air, temperature-controlled block on a Zeiss LSM5 Pascal confocal microscope (Carl Zeiss, Thornwood, NY). The distribution of GLP-1R-GFP was monitored in cells maintained in culture media at 37 C by acquiring micrographs every 4 sec for up to 5 min. For fixed cells microscopy, cells were treated as detailed in the figure legends, transferred to ice, rinsed in PBS, and fixed and permeabilized using a 2% paraformaldehyde-0.2% Triton X-100 solution for 30 min. Blocking was performed by incubating the cells for 1 h at room temperature in 4% FBS. Primary antibody (anticaveolin-1 rabbit polyclonal antibody), diluted 1:500 in PBS-1% BSA, was applied to the specimens for 12 h at 4 C, followed by three washes with the same buffer. Alexa635-tagged secondary antibody was diluted 1:500 and applied in the same buffer as the primary antibody for 2 h at room temperature. Coverslips were mounted for immunofluorescence microscopy, and analyzed with a Zeiss LSM5 Pascal confocal microscope with a 63x-oil immersion objective (Carl Zeiss).

Cell Fractionation and Western Blot Analysis
Triton X-100 soluble and insoluble cellular fractions were prepared from HEK293 cell lysates. Briefly, 48 h after transfection with 0.4 µg DNA/well of GFP-tagged GLP-1R or pcDNA3.1, cells were lysed on ice in lysis buffer (20 mM HEPES, pH 7.2; 100 mM NaCl, containing 1% Triton X-100, 10% glycerol, protease inhibitor cocktail set I, 500 µM sodium orthovanadate, and 5 mM NaF). The lysate was centrifuged at 16,000 x g for 10 min at 4 C, and the supernatant (Triton X-100 soluble fraction) was saved. The pellet was washed once with PBS at 4 C, resuspended in lysis buffer supplemented with 0.5% wt/vol sodium dodecyl sulfate, passed through a 30-gauge syringe, and then sonicated (Triton X-100 insoluble fraction). Equal amounts of protein (30 µg) from the Triton X-100 soluble and insoluble fractions were then resolved by SDS-PAGE and transferred onto nitrocellulose membrane (Amersham Biosciences, Arlington Heights, IL). Blots were then incubated overnight at 4 C with anti-GFP (1:1000) antibody diluted in 5% milk in TBS containing 0.2% Tween 20, extensively washed in TBS, and incubated in secondary antibody (1:2000; horseradish peroxidase-conjugated goat antimouse IgG), followed by detection of by chemiluminescence and autoradiography. Autoradiographs were digitized, and subsequent analyses were performed with Image J software (National Institutes of Health).

Triton X-100-Based Isolation of Caveolae
Caveolae were purified based on an isolation protocol of Leclerc et al. (29) adapted from methods of Lisanti and colleagues (28). Briefly, HEK293 cells stably transfected with GLP-1R-GFP were grown to confluence. Cells were lysed in an ice-cold 2-[N-morpholino]ethanesulfonic acid (MES)-buffered solution (25 mM MES, pH 6.5; 150 mM NaCl) containing 1% Triton X-100, and protease inhibitor cocktail. After homogenization with 10 strokes of a Dounce homogenizer (Kontes Glass Co., Vineland, NJ), the extracts were adjusted to 45% sucrose with MES-buffered solution (lacking Triton X-100) and distributed at the bottom of ultracentrifuge tubes. The samples were then overlaid with 4 ml 35% sucrose followed by 4 ml of a 5% sucrose cushion and centrifuged at 39,000 rpm for 16–20 h in an SW41 rotor (Beckman Instruments, Palo Alto, CA). Fractions (1 ml) were then collected and subjected to SDS-PAGE.

Immunoprecipitation
Cells were lysed with 200 µl PBS containing 1 mM CaCl2, 1% vol/vol Triton X-100, 0.5% wt/vol sodium dodecyl sulfate, and protease inhibitor cocktail mix. The lysate was centrifuged at 14,000 x g for 15 min at 4 C, and 150 µl supernatant was rocked overnight at 4 C with 25 µl anti-GFP agarose conjugate (Santa Cruz Biotechnology). The pellet was washed once with lysis buffer and four times with PBS (pellet centrifuged at 1000 x g for 30 sec). The pellet was then resuspended in 2x Laemmli buffer, and proteins were resolved on 10% SDS-PAGE and transferred onto nitrocellulose membrane (Amersham Biosciences). Blots were then incubated overnight at 4 C with either anti-GFP (1:1000) or anti-caveolin-1 (1:500) antibodies diluted in 5% milk in TBS containing 0.2% Tween 20. In an alternative approach, cell lysates were preincubated with 10 µl anticaveolin-1 antibody for 90 min at 4 C, and then 20 µl Protein A-agarose (Santa Cruz Biotechnology) was added and then left overnight on a rocker at 4 C. The remainder of the procedure was then performed as described above.

Statistics
Data are presented as means ± SEM, where n indicates the number of independent experiments. Statistical analysis was performed using two-tailed Student’s t test, as indicated in the figure legends.


   ACKNOWLEDGMENTS
 
We thank the following colleagues for kindly providing reagents: Dr. Orson Moe (University of Texas, Dallas TX) for K44A-dynamin, Dr. Ferruccio Galbiati (University of Pittsburgh, Pittsburgh PA) for caveolin-1 constructs, Dr. Riccardo Perfetti (Cedars-Sinai Medical Center, Los Angeles CA) for GLP-1R cDNA, Dr. Marc Caron (Duke University, Durham NC) for ß-Arr2-GFP, and Dr. Susumu Seino (Kobe University, Kobe, Japan) for MIN6 cells.


   FOOTNOTES
 
This work was supported, in part, by Grants DK046204 and DK07052 from the National Institute of Diabetes and Digestive and Kidney Diseases.

Disclosure summary: The authors have nothing to declare.

First Published Online August 24, 2006

Abbreviations: AT1, Angiotensin II type 1; ß-Arr2-GFP, ß-arrestin2-GFP; EGF, epidermal growth factor; ETA, endothelin A; FBS, fetal bovine serum; GFP, green fluorescent protein; GLP-1, glucagon-like peptide-1(7–36); GLP-1R, glucagon-like peptide 1 receptor; GPCR, G protein-coupled receptor; HEK293, human embryonic kidney; IBMX, 3-isobutyl-1-methylxanthine; MES, 2-[N-morpholino]ethanesulfonic acid; PI3K, phosphatidylinositol 3-kinase; PTH1R, PTH 1 receptor; TBS, Tris-buffered saline.

Received for publication April 26, 2006. Accepted for publication August 18, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Thorens B 1992 Expression cloning of the pancreatic ß cell receptor for the gluco-incretin hormone glucagon-like peptide 1. Proc Natl Acad Sci USA 89:8641–8645[Abstract/Free Full Text]
  2. Thorens B, Porret A, Buhler L, Deng SP, Morel P, Widmann C 1993 Cloning and functional expression of the human islet GLP-1 receptor. Demonstration that exendin-4 is an agonist and exendin-(9–39) an antagonist of the receptor. Diabetes 42:1678–1682[Abstract]
  3. Eng J, Kleinman WA, Singh L, Singh G, Raufman JP 1992 Isolation and characterization of exendin-4, an exendin-3 analogue, from Heloderma suspectum venom. Further evidence for an exendin receptor on dispersed acini from guinea pig pancreas. J Biol Chem 267:7402–7405[Abstract/Free Full Text]
  4. Drucker DJ 2003 Glucagon-like peptide-1 and the islet ß-cell: augmentation of cell proliferation and inhibition of apoptosis. Endocrinology 144:5145–5148[Free Full Text]
  5. Brubaker PL, Drucker DJ 2004 Minireview: glucagon-like peptides regulate cell proliferation and apoptosis in the pancreas, gut, and central nervous system. Endocrinology 145:2653–2659[Abstract/Free Full Text]
  6. Perry T, Greig NH 2003 The glucagon-like peptides: a double-edged therapeutic sword? Trends Pharmacol Sci 24:377–383[CrossRef][Medline]
  7. Drucker DJ, Philippe J, Mojsov S, Chick WL, Habener JF 1987 Glucagon-like peptide I stimulates insulin gene expression and increases cyclic AMP levels in a rat islet cell line. Proc Natl Acad Sci USA 84:3434–3438[Abstract/Free Full Text]
  8. Widmann C, Burki E, Dolci W, Thorens B 1994 Signal transduction by the cloned glucagon-like peptide-1 receptor: comparison with signaling by the endogenous receptors of ß cell lines. Mol Pharmacol 45:1029–1035[Abstract]
  9. Arnette D, Gibson TB, Lawrence MC, January B, Khoo S, McGlynn K, Vanderbilt CA, Cobb MH 2003 Regulation of ERK1 and ERK2 by glucose and peptide hormones in pancreatic ß cells. J Biol Chem 278:32517–32525[Abstract/Free Full Text]
  10. Gomez E, Pritchard C, Herbert TP 2002 cAMP-dependent protein kinase and Ca2+ influx through L-type voltage-gated calcium channels mediate Raf-independent activation of extracellular regulated kinase in response to glucagon-like peptide-1 in pancreatic ß-cells. J Biol Chem 277:48146–48151[Abstract/Free Full Text]
  11. Briaud I, Lingohr MK, Dickson LM, Wrede CE, Rhodes CJ 2003 Differential activation mechanisms of Erk-1/2 and p70(S6K) by glucose in pancreatic ß-cells. Diabetes 52:974–983[Abstract/Free Full Text]
  12. Buteau J, Foisy S, Joly E, Prentki M 2003 Glucagon-like peptide 1 induces pancreatic ß-cell proliferation via transactivation of the epidermal growth factor receptor. Diabetes 52:124–132[Abstract/Free Full Text]
  13. Widmann C, Dolci W, Thorens B 1996 Heterologous desensitization of the glucagon-like peptide-1 receptor by phorbol esters requires phosphorylation of the cytoplasmic tail at four different sites. J Biol Chem 271:19957–19963[Abstract/Free Full Text]
  14. Widmann C, Dolci W, Thorens B 1997 Internalization and homologous desensitization of the GLP-1 receptor depend on phosphorylation of the receptor carboxyl tail at the same three sites. Mol Endocrinol 11:1094–1102[Abstract/Free Full Text]
  15. Widmann C, Dolci W, Thorens B 1995 Agonist-induced internalization and recycling of the glucagon-like peptide-1 receptor in transfected fibroblasts and in insulinomas. Biochem J 310:203–214[Medline]
  16. Wang Y, Perfetti R, Greig NH, Holloway HW, DeOre KA, Montrose-Rafizadeh C, Elahi D, Egan JM 1997 Glucagon-like peptide-1 can reverse the age-related decline in glucose tolerance in rats. J Clin Invest 99:2883–2889[Medline]
  17. Baggio LL, Kim JG, Drucker DJ 2004 Chronic exposure to GLP-1R agonists promotes homologous GLP-1 receptor desensitization in vitro but does not attenuate GLP-1R-dependent glucose homeostasis in vivo. Diabetes 53(Suppl 3):S205–S214
  18. Luttrell LM, Lefkowitz RJ 2002 The role of ß-arrestins in the termination and transduction of G-protein-coupled receptor signals. J Cell Sci 115:455–465[Abstract/Free Full Text]
  19. Chini B, Parenti M 2004 G-protein coupled receptors in lipid rafts and caveolae: how, when and why do they go there? J Mol Endocrinol 32:325–338[Abstract]
  20. Pelkmans L, Kartenbeck J, Helenius A 2001 Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nat Cell Biol 3:473–483[CrossRef][Medline]
  21. Chun M, Liyanage UK, Lisanti MP, Lodish HF 1994 Signal transduction of a G protein-coupled receptor in caveolae: colocalization of endothelin and its receptor with caveolin. Proc Natl Acad Sci USA 91:11728–11732[Abstract/Free Full Text]
  22. Schwencke C, Okumura S, Yamamoto M, Geng YJ, Ishikawa Y 1999 Colocalization of ß-adrenergic receptors and caveolin within the plasma membrane. J Cell Biochem 75:64–72[CrossRef][Medline]
  23. Rybin VO, Xu X, Lisanti MP, Steinberg SF 2000 Differential targeting of ß-adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling pathway. J Biol Chem 275:41447–41457[Abstract/Free Full Text]
  24. Okamoto Y, Ninomiya H, Miwa S, Masaki T 2000 Cholesterol oxidation switches the internalization pathway of endothelin receptor type A from caveolae to clathrin-coated pits in Chinese hamster ovary cells. J Biol Chem 275:6439–6446[Abstract/Free Full Text]
  25. Couet J, Li S, Okamoto T, Ikezu T, Lisanti MP 1997 Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins. J Biol Chem 272:6525–6533[Abstract/Free Full Text]
  26. Okamoto T, Schlegel A, Scherer PE, Lisanti MP 1998 Caveolins, a family of scaffolding proteins for organizing "preassembled signaling complexes" at the plasma membrane. J Biol Chem 273:5419–5422[Free Full Text]
  27. Minami K, Yano H, Miki T, Nagashima K, Wang CZ, Tanaka H, Miyazaki JI, Seino S 2000 Insulin secretion and differential gene expression in glucose-responsive and -unresponsive MIN6 sublines. Am J Physiol Endocrinol Metab 279:E773–E781
  28. Song KS, Li S, Okamoto T, Quilliam LA, Sargiacomo M, Lisanti MP 1996 Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free purification of caveolae microdomains. J Biol Chem 271:9690–9697[Abstract/Free Full Text]
  29. Leclerc PC, Auger-Messier M, Lanctot PM, Escher E, Leduc R, Guillemette G 2002 A polyaromatic caveolin-binding-like motif in the cytoplasmic tail of the type 1 receptor for angiotensin II plays an important role in receptor trafficking and signaling. Endocrinology 143:4702–4710[Abstract/Free Full Text]
  30. Lee H, Park DS, Razani B, Russell RG, Pestell RG, Lisanti MP 2002 Caveolin-1 mutations (P132L and null) and the pathogenesis of breast cancer: caveolin-1 (P132L) behaves in a dominant-negative manner and caveolin-1 (–/–) null mice show mammary epithelial cell hyperplasia. Am J Pathol 161:1357–1369[Abstract/Free Full Text]
  31. Ferrari SL, Behar V, Chorev M, Rosenblatt M, Bisello A 1999 Endocytosis of ligand-human parathyroid hormone receptor 1 complexes is protein kinase C-dependent and involves ß-arrestin2. Real-time monitoring by fluorescence microscopy. J Biol Chem 274:29968–29975[Abstract/Free Full Text]
  32. Ferrari SL, Bisello A 2001 Cellular distribution of constitutively active mutant parathyroid hormone (PTH)/PTH-related protein receptors and regulation of cyclic adenosine 3',5'-monophosphate signaling by ß-arrestin2. Mol Endocrinol 15:149–163[Abstract/Free Full Text]
  33. Westover EJ, Covey DF, Brockman HL, Brown RE, Pike LJ 2003 Cholesterol depletion results in site-specific increases in epidermal growth factor receptor phosphorylation due to membrane level effects. Studies with cholesterol enantiomers. J Biol Chem 278:51125–51133[Abstract/Free Full Text]
  34. Pike LJ, Casey L 2002 Cholesterol levels modulate EGF receptor-mediated signaling by altering receptor function and trafficking. Biochemistry 41:10315–10322[CrossRef][Medline]
  35. Wyse BD, Prior IA, Qian H, Morrow IC, Nixon S, Muncke C, Kurzchalia TV, Thomas WG, Parton RG, Hancock JF 2003 Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane. J Biol Chem 278:23738–23746[Abstract/Free Full Text]
  36. Yamaguchi T, Murata Y, Fujiyoshi Y, Doi T 2003 Regulated interaction of endothelin B receptor with caveolin-1. Eur J Biochem 270:1816–1827[Medline]
  37. Nystrom FH, Chen H, Cong L-N, Li Y, Quon MJ 1999 Caveolin-1 interacts with the insulin receptor and can differentially modulate insulin signaling in transfected Cos-7 cells and rat adipose cells. Mol Endocrinol 13:2013–2024[Abstract/Free Full Text]
  38. Fehmann HC, Jiang J, Pitt D, Schweinfurth J, Goke B 1996 Ligand-induced regulation of glucagon-like peptide-I receptor function and expression in insulin-secreting ß cells. Pancreas 13:273–282[Medline]
  39. Gromada J, Dissing S, Rorsman P 1996 Desensitization of glucagon-like peptide 1 receptors in insulin-secreting ß TC3 cells: role of PKA-independent mechanisms. Br J Pharmacol 118:769–775
  40. Widmann C, Dolci W, Thorens B 1996 Desensitization and phosphorylation of the glucagon-like peptide-1 (GLP-1) receptor by GLP-1 and 4-phorbol 12-myristate 13-acetate. Mol Endocrinol 10:62–75[Abstract]
  41. Fehmann HC, Pracht A, Goke B 1998 High-level expression of the GLP-I receptor results in receptor desensitization. Pancreas 17:309–314[Medline]
  42. Estall JL, Yusta B, Drucker DJ 2004 Lipid raft-dependent glucagon-like peptide-2 receptor trafficking occurs independently of agonist-induced desensitization. Mol Biol Cell 15:3673–3687[Abstract/Free Full Text]
  43. Kifor O, Diaz R, Butters R, Kifor I, Brown EM 1998 The calcium-sensing receptor is localized in caveolin-rich plasma membrane domains of bovine parathyroid cells. J Biol Chem 273:21708–21713[Abstract/Free Full Text]
  44. Kifor O, Kifor I, Moore Jr FD, Butters Jr RR, Cantor T, Gao P, Brown EM 2003 Decreased expression of caveolin-1 and altered regulation of mitogen-activated protein kinase in cultured bovine parathyroid cells and human parathyroid adenomas. J Clin Endocrinol Metab 88:4455–4464[Abstract/Free Full Text]
  45. Feron O, Han X, Kelly RA 1999 Muscarinic cholinergic signaling in cardiac myocytes: dynamic targeting of M2AChR to sarcolemmal caveolae and eNOS activation. Life Sci 64:471–477[CrossRef][Medline]
  46. Roettger BF, Rentsch RU, Pinon D, Holicky E, Hadac E, Larkin JM, Miller LJ 1995 Dual pathways of internalization of the cholecystokinin receptor. J Cell Biol 128:1029–1041[Abstract/Free Full Text]
  47. Buteau J, Roduit R, Susini S, Prentki M 1999 Glucagon-like peptide-1 promotes DNA synthesis, activates phosphatidylinositol 3-kinase and increases transcription factor pancreatic and duodenal homeobox gene 1 (PDX-1) DNA binding activity in ß (INS-1)-cells. Diabetologia 42:856–864[CrossRef][Medline]
  48. Buteau J, Foisy B, Rhodes CJ, Carpenter L, Biden TJ, Prentki M 2001 Protein kinase C{zeta} activation mediates glucagon-like peptide-1-induced pancreatic ß-cell proliferation. Diabetes 50:2237–2243[Abstract/Free Full Text]
  49. Wang Q, Li L, Xu E, Wong V, Rhodes C, Brubaker PL 2004 Glucagon-like peptide-1 regulates proliferation and apoptosis via activation of protein kinase B in pancreatic INS-1 ß cells. Diabetologia 47:478–487[CrossRef][Medline]
  50. Jorgensen R, Martini L, Schwartz TW, Elling CE 2005 Characterization of glucagon-like peptide-1 receptor ß-arrestin 2 interaction: a high-affinity receptor phenotype. Mol Endocrinol 19:812–823[Abstract/Free Full Text]
  51. Wei H, Ahn S, Shenoy SK, Karnik SS, Hunyady L, Luttrell LM, Lefkowitz RJ 2003 Independent ß-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2. Proc Natl Acad Sci USA 100:10782–10787[Abstract/Free Full Text]
  52. Syme CA, Friedman PA, Bisello A 2005 Parathyroid hormone receptor trafficking contributes to the activation of extracellular signal-regulated kinases but is not required for regulation of cAMP signaling. J Biol Chem 280:11281–11288[Abstract/Free Full Text]
  53. Sneddon WB, Syme CA, Bisello A, Magyar CE, Rochdi MD, Parent J-L, Weinman EJ, Abou-Samra AB, Friedman PA 2003 Activation-independent parathyroid hormone receptor internalization is regulated by NHERF1 (EBP50). J Biol Chem 278:43787–43796[Abstract/Free Full Text]
  54. Behar V, Pines M, Nakamoto C, Greenberg Z, Bisello A, Stueckle SM, Bessalle R, Usdin TB, Chorev M, Rosenblatt M, Suva LJ 1996 The human PTH2 receptor: binding and signal transduction properties of the stably expressed recombinant receptor. Endocrinology 137:2748–2757[Abstract]
  55. Salomon Y, Londos C, Rodbell M 1974 A highly sensitive adenylate cyclase assay. Anal Biochem 58:541–548[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
N. Sonoda, T. Imamura, T. Yoshizaki, J. L. Babendure, J.-C. Lu, and J. M. Olefsky
{beta}-Arrestin-1 mediates glucagon-like peptide-1 signaling to insulin secretion in cultured pancreatic {beta} cells
PNAS, May 6, 2008; 105(18): 6614 - 6619.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Alioua, R. Lu, Y. Kumar, M. Eghbali, P. Kundu, L. Toro, and E. Stefani
Slo1 Caveolin-binding Motif, a Mechanism of Caveolin-1-Slo1 Interaction Regulating Slo1 Surface Expression
J. Biol. Chem., February 22, 2008; 283(8): 4808 - 4817.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
R. Jorgensen, V. Kubale, M. Vrecl, T. W. Schwartz, and C. E. Elling
Oxyntomodulin Differentially Affects Glucagon-Like Peptide-1 Receptor beta-Arrestin Recruitment and Signaling through G{alpha}
J. Pharmacol. Exp. Ther., July 1, 2007; 322(1): 148 - 154.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
20/12/3400    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Syme, C. A.
Right arrow