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Molecular Endocrinology, doi:10.1210/me.2004-0275
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Molecular Endocrinology 19 (2): 474-490
Copyright © 2005 by The Endocrine Society

Regulation of Corticotropin-Releasing Hormone Receptor Type 1{alpha} Signaling: Structural Determinants for G Protein-Coupled Receptor Kinase-Mediated Phosphorylation and Agonist-Mediated Desensitization

Thalia Teli, Danijela Markovic, Michael A. Levine, Edward W. Hillhouse and Dimitris K. Grammatopoulos

Sir Quinton Hazell Molecular Medicine Research Centre (T.T., D.M., D.K.G.), Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom; Division of Paediatrics (M.A.L.), The Children’s Hospital of The Cleveland Clinic Foundation, Cleveland, Ohio 44195; and The Leeds Institute of Health (E.W.H.), Genetics and Therapeutics, University of Leeds, Leeds LS2 9NL, United Kingdom

Address all correspondence and requests for reprints to: Dr. D.Grammatopoulos, Sir Quinton Hazell Molecular Medicine Research Centre, Department of Biological Sciences, The University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom. E-mail: d.grammatopoulos{at}warwick.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Attenuation of CRH receptor type 1 (CRH-R1) signaling activity might involve desensitization and uncoupling of CRH-R1 from intracellular effectors. We investigated the desensitization of native CRH-R in human myometrial cells from pregnant women and recombinant CRH-R1{alpha} stably overexpressed in human embryonic kidney (HEK) 293 cells. In both cell types, CRH-R1-mediated adenylyl cyclase activation was susceptible to homologous desensitization induced by pretreatment with high concentrations of CRH. Time course studies showed half-maximal desensitization occurring after approximately 40 min of pretreatment and full recovery of CRH-R1{alpha} functional response within 2 h of removal of CRH pretreatment. In HEK 293 cells, desensitization of CRH-R1{alpha} was associated with receptor phosphorylation and subsequent endocytosis. To analyze the mechanism leading to CRH-R1{alpha} desensitization, we overexpressed a truncated ß-arrestin (319–418) and performed coimmunoprecipitation and G protein-coupled receptor kinase (GRK) translocation studies. We found that GRK3 and GRK6 are the main isoforms that interact with CRH-R1{alpha}, and that recruitment of GRK3 requires Gß{gamma}-subunits as well as ß-arrestin. Site-directed mutagenesis of Ser and Thr residues in the CRH-R1{alpha} C terminus, identified Thr399 as important for GRK-induced receptor phosphorylation and desensitization.

We conclude that homologous desensitization of CRH-R1{alpha} involves the coordinated action of multiple GRK isoforms, Gß {gamma} dimers and ß-arrestin. Based on our identification of key amino acid(s) for GRK-dependent phosphorylation, we demonstrate the importance of the CRH-R1{alpha} carboxyl tail for regulation of receptor activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TWO FAMILIES OF G protein-coupled receptors (GPCRs), termed CRH receptors R1 and R2, mediate actions of CRH and CRH-like agonists [urocortins (UCNs), sauvagine, and urotensin I] in target tissues (1, 2). The CRH-R1 exists as multiple isoforms (3), arising from alternative exon splicing. Targeted disruption of the gene encoding CRH-R1 in mice provided direct evidence that this receptor mediates CRH actions in response to stressful stimuli through activation of pituitary corticotropes and ACTH secretion (4). The CRH-R1 is widely expressed in many tissues (5, 6, 7), which suggests additional role(s) for CRH-R1 agonists. In most cells, binding of CRH or UCN to CRH-R1 leads to activation of adenylyl cyclase (AC), and increased protein kinase A (PKA) activity. Further signal diversity arises from the ability of CRH-R1 to activate multiple G proteins (8, 9, 10), in a tissue-specific manner, which results in generation of various second messengers (3).

During human pregnancy, the CRH-R1-activating agonists, CRH and UCN, are produced by the placenta and other intrauterine tissues (11, 12). Placental CRH production increases exponentially as pregnancy progresses toward labor, and it has been proposed that CRH might act as a placental clock regulating the length of human gestation (13). The precise biological role of placental CRH and CRH-related peptides during pregnancy and labor remains unknown, but the human myometrium appears to be a major target of their actions (14). Extensive in vitro studies in our laboratory suggest that, during pregnancy, CRH targets the human myometrium and initiates signaling cascades, which are primarily involved in maintaining uterine quiescence. In myometrial smooth muscle cells, CRH activation of CRH-R1{alpha} (the most potent CRH-R1 isoform) can stimulate production of cAMP and cyclic GMP (15, 16) and induces up-regulation of nitric oxide synthase (16), pathways leading to inhibition of myosin light chain phosphorylation and increased myometrial relaxation (17). By contrast, UCN activation of myometrial CRH-R1{alpha} receptors stimulates also other signaling cascades, such as the protein kinase C (PKC)/ERK, which leads to increased myometrial contractility (18). These signaling pathways are modified by a self-regulatory mechanism in which PKA-induced phosphorylation of Ser301 of the CRH-R1{alpha} results in attenuation of ERK activity (19). This mechanism could account for the ability of CRH-R1{alpha} agonists to exert the contrasting roles of maintaining myometrial relaxation during preterm pregnancy and stimulating contractility (20) during labor.

Little is known about the cellular mechanisms controlling CRH-R1{alpha} activity in myometrial and other target cells. The signaling activity of most GPCRs is attenuated using phosphorylation by protein kinases [e.g. PKA, PKC, and G protein-coupled receptor kinases (GRKs)] that allows interaction with arrestins leading to receptor desensitization and uncoupling from G proteins (21) followed by receptor internalization (22). In various cell lines, such as mouse fibroblasts and the Y79 human retinoblastoma cell line (23, 24, 25, 26), the CRH-R1 appears to undergo homologous desensitization after exposure to high concentrations of CRH via a process that is PKA independent but requires GRK3. This process appears to be important in the regulation of myometrial responsiveness to external stimuli during pregnancy and labor (27) and might contribute to the signaling uncoupling of myometrial CRH receptors, observed at the end of pregnancy (15).

In this study, we analyzed native CRH-R1{alpha} receptors in human myometrial smooth muscle cells and recombinant CRH-R1{alpha} stably expressed in human embryonic kidney (HEK) 293 cells to investigate homologous desensitization of the CRH-R1{alpha} after CRH binding and receptor activation, and to identify potential receptor domains that are involved in this process.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Desensitization Characteristics of CRH Signaling in Myocytes from Pregnant Women and HEK 293-R1{alpha} Cells
To determine whether human myometrial CRH-R signaling is susceptible to homologous desensitization, we assessed the effect of human/rat CRH pretreatment on the maximal CRH-induced adenylyl cyclase activation and intracellular cAMP accumulation. Pretreatment of cultured myocytes from pregnant women, for 30 min with increasing concentrations of CRH (10–100 nM) resulted in a dose-dependent attenuation of the myometrial cell cAMP response to 100 nM CRH (Fig. 1AGo); inhibition reached 58 ± 5% with 100 nM CRH pretreatment.



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  		Fig. 1. CRH-R Desensitization Characteristics in Native and Overexpression Cellular Systems

A, Effect of CRH pretreatment on CRH-induced cAMP production in human myometrial cells from pregnant women. Cells were pretreated with increasing concentrations of CRH (1–100 nM) for 30 min to induce desensitization. After extensive washing, dose-dependent (10 pM to 100 nM) response of cAMP to a second CRH stimulus (15 min) was determined. Data represent the mean ± SEM of two estimations from three independent experiments. *, P < 0.05 compared with cells without CRH pretreatment. Basal cAMP levels 3 ± 2.2 pmol/ml/106 cells. B, Detection of CRH-R1{alpha} expression in st.293-R1{alpha} vs. untransfected cells by Western blot analysis. Cell lysates from HEK 293 cells stably expressing wild-type (w.t) CRH-R1{alpha} receptors or untransfected HEK 293 cells, were fractionated on SDS-PAGE, and subjected to immunoblotting with a specific CRH-R1/2 antibody as described in Materials and Methods. Identical results were obtained from two independent transfection experiments. C, Effect of CRH pretreatment on CRH-induced Gs{alpha}-protein activation in membranes prepared from st.293-R1{alpha} cells. Membranes from CRH-pretreated cells were incubated with GTP-AA in the presence or absence of CRH (100 nM) followed by UV cross-linking and immunoprecipitation of the Gs{alpha}-subunits using specific antibodies. Proteins were resolved on SDS-PAGE gels, followed by autoradiography and densitometry scanning to quantify agonist-induced photolabeling of Gs{alpha}-subunits.

 
Because CRH effects in cultured myometrial cells from pregnant women are primarily mediated via the CRH-R1{alpha} (15, 16), this receptor was stably expressed in HEK 293 cells (st.293-R1{alpha} cells). Successful expression of CRH-R1{alpha} in these cells was confirmed by immunoblotting using a specific CRH-R1/2 antibody (Fig. 1BGo) and the mechanism underlying desensitization of CRH-R1{alpha} responsiveness after an initial stimulation with CRH was investigated. Pretreatment of st.293-R1{alpha} cells for 30min with CRH at concentrations greater than 10 nM resulted in a 38 ± 7% reduction in subsequent CRH (100 nM)-induced cAMP response (data not shown). Subsequent experiments with the G{alpha}-protein photoaffinity label 32P-GTP-AA showed that in CRH-pretreatment of st.293-R1{alpha} cells, reduced coupling of CRH-R1{alpha} to Gs{alpha}. As shown in Fig. 1CGo, pretreatment of st.293-R1{alpha} cells with 100 nM CRH for 30 min, led to a 53 ± 10% reduction in subsequent CRH-induced Gs{alpha}-protein activation, without altering expression of Gs{alpha} protein.

Homologous desensitization of the CRH-R1{alpha} was also found to be time dependent; agonist-induced desensitization was apparent after 15 min of preexposure to 100 nM CRH and maximal attenuation of the CRH-induced AC activation (75 ± 9% reduction) was observed when cells were pretreated for 3 h before the second 15 min CRH stimulus (Fig. 2AGo). In a different set of experiments, the recovery kinetics of the CRH-R1{alpha} response was also investigated. After pretreatment of st.293-R1{alpha} cells with 100 nM CRH for 3 h (to induce maximal receptor desensitization), the cells were washed to remove ligand and allowed to rest for varying intervals before addition of 100 nM CRH for 15 min. A 50% recovery of the CRH responsiveness was apparent after a 60-min resting period, and full recovery was achieved with cells cultured for 2 h before addition of CRH (Fig. 2BGo). At all time intervals, basal and forskolin-stimulated cAMP levels were not different between treated and untreated cells (data not shown).



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  		Fig. 2. Desensitization Characteristics of the CRH-R1{alpha} Receptor

A, Time course of CRH-R1{alpha} desensitization in st.293-R1{alpha} cells. CRH pretreated cells (100 nM for various time intervals) were stimulated with 100 nM CRH for 15min and the subsequent cAMP response was determined. B, Time course of CRH-R1{alpha} resensitization in st.293-R1{alpha} cells. CRH pretreated cells (100 nM for 3 h to induce maximal receptor desensitization) were briefly washed, and allowed to recover for various time intervals before a second 100 nM CRH stimulus for 15 min and measurement of the subsequent cAMP accumulation by RIA. Data represent the mean ± SEM of two estimations from three independent experiments. *, P < 0.05 compared with cells without CRH pretreatment (nondesensitized); +, P < 0.05 compared with maximally desensitized cells. Basal cAMP levels 1.5 ± 0.4 pmol/ml/2 x 105 cells.

 
To assess whether homologous desensitization was associated with CRH-R1{alpha} internalization, we used indirect immunofluorescence with a specific CRH-R antibody to monitor distribution of the receptor after addition of CRH for various time intervals. In the absence of CRH, CRH-R1{alpha} receptors were exclusively localized on the cell surface of st.293-R1{alpha} cells; treatment of cells with concentrations of CRH greater than 10 nM for 45 min elicited a redistribution of cellular immunostaining, indicative of receptor internalization (Fig. 3AGo). Furthermore, the loss of CRH-R1{alpha} from the cell surface, was dependent upon the time of CRH treatment, with a significant increase in CRH-R1{alpha} signal inside the cell within 30 min of CRH treatment and, intense receptor signal was observed in the perinuclear region after 60 min of CRH treatment (Fig. 3BGo). No significant staining was observed in non transfected HEK 293 cells (data not shown).



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  		Fig. 3. Agonist-Concentration Dependency (A) and Time Course (B) of CRH-R1{alpha} Internalization in HEK 293 Cells: Visualization by Confocal Microscopy

st.293-R1{alpha} cells were grown in coverslips and after exposure to CRH (0.1–100 nM) for various time periods, CRH-R1{alpha} distribution was monitored over the ensuing time period by indirect immunofluorescence using CRH-R1-specific antiserum and Alexa-Fluor 594 secondary antibody. Identical results were obtained from four independent experiments.

 
Role of Protein Kinases and ß-Arrestin in CRH-R1{alpha} Desensitization
For most, but not all, GPCRs, one of the initial steps in the process of receptor desensitization is receptor phosphorylation by protein kinases (28). We therefore analyzed, in vitro phosphorylation of CRH-R1{alpha} in 32P-labeled st.293-R1{alpha} cells as previously described (19). These experiments showed that CRH induced incorporation of 32P into a 45- to 50-kDa protein that was immunoprecipitated by specific CRH-R1/2 Abs, confirming that the receptor was indeed a target of CRH-induced phosphorylation (Fig. 4AGo).



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  		Fig. 4. In Vitro Phosphorylation (A) and Homologous Desensitization (B) Characteristics of Wild-Type (w.t) and (S301E) Mutant CRH-R1{alpha} Receptors

A, HEK 293 stably expressing w.t and (S301E)CRH-R1{alpha}, were treated with 100 nM CRH for 30 min, in the presence of 32P. Phosphorylated CRH-Rs were solubilized, immunoprecipitated with specific CRH-R1/2 antibodies, fractionated on SDS-PAGE, and were subjected to autoradiography (–70 C, 10–14 d) using intensifying screens. identical results were obtained from three independent experiments. B, HEK 293 cells stably expressing w.t and mutant CRH-R1{alpha} receptors were pretreated with or without CRH (100 nM for 60 min) to induce desensitization. After extensive washing, the cAMP response after a second 100 nM CRH stimulus for 15 min was determined. Data represent the mean ± SEM of two estimations from three independent experiments. *, P < 0.05 compared with cells without CRH pretreatment. +, Basal cAMP levels 2.3 ± 0.5 pmol/ml/2 x105 cells.

 
Phosphorylation of GPCRs can involve a variety of protein kinases, including PKA, PKC, and GPCR kinases (GRKs). The role of PKA in CRH-R1{alpha} desensitization process was investigated in detail, by pretreatment of st.293-R1{alpha} cells with 10 µM forskolin (to stimulate directly AC and PKA activity) for various time intervals, before a washing step and stimulation with 100 nM CRH for 15 min. Measurement of cAMP production showed that pretreatment of the cells with forskolin for up to 3 h did not alter CRH-induced adenylyl cyclase (AC) activation (Table 1Go). These results, in the context of our previous findings (19) suggested that PKA-induced phosphorylation of CRH-R1{alpha} does not lead to desensitization. To conclusively demonstrate this, we used the PKA-phosphorylation-resistant mutant receptor (S301E)CRH-R1{alpha} that showed similar to the wild-type CRH-R1{alpha}, potency in activating the AC and ERK signaling pathways in HEK 293 cells. When stably expressed in HEK 293 cells, this mutant receptor exhibited significantly reduced (by 25–35%) receptor phosphorylation in response to CRH treatment (100 nM for 15 min), compared with the wild-type CRH-R1{alpha} (Fig. 4AGo) probably reflecting the absence of PKA-induced phosphorylation. Most importantly, the mutant receptor exhibited homologous desensitization responses to CRH pretreatment that were comparable to that of the wild-type receptor (Fig. 4BGo), confirming that PKA was not involved in CRH-R1{alpha} homologous desensitization.


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  		Table 1. Effect of Adenylyl Cyclase Preactivation by Forskolin on CRH-Induced cAMP Accumulation in st.293-R1{alpha} Cells

 
The role of GRKs on CRH-induced CRH-R1{alpha} desensitization was tested by using the nonselective GRK inhibitors heparin and Zn2+. Treatment of streptolysin O (SLO)-permeabilized st.293-R1{alpha} cells with heparin (1 µM, 5 min) led to a near complete abrogation of CRH-induced CRH-R1{alpha} desensitization (Fig. 5AGo). In agreement with this, heparin treatment significantly reduced CRH-induced receptor phosphorylation in st.293-R1{alpha} cells, suggesting that heparin-sensitive GRKs are mediating CRH-R1{alpha} phosphorylation (Fig. 5BGo). Similar results were obtained when st.293-R1{alpha} cells were exposed to 100 µM ZnCl2 (data not shown).



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  		Fig. 5. Effect of Heparin Treatment on CRH-Induced (A) Homologous Desensitization and (B) Phosphorylation of CRH-R1{alpha} in HEK 293 Cells

st.293-R1{alpha} cells were permeabilized with SLO (10 µg/ml), and exposed to heparin (1 µM, 1 min) before pretreatment with 100 nM CRH for 60 min. A, CRH-R1{alpha} desensitization was determined by measurement of cAMP production in response to stimulation with 100 nM of CRH for 15 min. Data represent the mean ± SEM of two estimations from two independent experiments. *, P < 0.05 compared with cells without CRH pretreatment. Basal cAMP levels 2.5 ± 0.7 pmol/ml/2 x 105 cells. B, Representative autoradiograph (top panel) of CRH-induced receptor phosphorylation in the presence of 32P, determined by cell lysate solubilization, immunoprecipitation of CRH-Rs with specific CRH-R1/2 antibodies, fractionation on SDS-PAGE, and autoradiography (–70 C, 10–14 d) using intensifying screens. This was followed by densitometry scanning to quantify agonist-induced receptor phosphorylation. The data in bottom panel represent the mean ± SEM of three independent experiments. *, P < 0.05 compared with CRH-untreated (basal) cells; +, P < 0.05 compared with heparin-untreated cells.

 
To investigate whether GRK-mediated CRH-R1{alpha} desensitization requires binding of ß-arrestin to the phosphorylated receptor, we used a ß-arrestin dominant-negative (ß-arrestin- (319–418), that binds to clathrin but not to GPCRs, and inhibits desensitization and internalization (29). In st.293-R1{alpha} cells, overexpression of ß-arrestin- (319–418) led to a 60 ± 5% inhibition of CRH-R1{alpha} desensitization induced by pretreatment with 100 nM CRH for 60 min (Fig. 6Go), suggesting that CRH-R1{alpha} homologous desensitization is a GRK and ß-arrestin-dependent pathway.



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  		Fig. 6. Effect of ß-Arrestin (319–418) Mutant on the Desensitization of CRH-R1{alpha} Stably Expressed in HEK 293 Cells

Eight micrograms of empty pcDNA3 vector (control) or ß-arrestin (319–418) in pcDNA3 were transfected in st.293-R1{alpha} cells and the effect on CRH-R1{alpha} homologous desensitization (induced by pretreatment with 100 nM CRH for 60 min) was determined, by measurement of CRH-induced cAMP production. Data represent the mean ± SEM of two estimations from three independent experiments. *, P < 0.05 compared with cells without CRH pretreatment. +, P < 0.05 compared with control cells. Basal cAMP levels 1.8 ± 0.8 pmol/ml/2 x 105 cells.

 
Specific GRK Isoforms and the Role of ß{gamma}-Subunits in CRH-R1{alpha} Desensitization
In view of the involvement of GRKs in the phosphorylation and subsequent desensitization of the CRH-R1{alpha} receptor, we investigated the GRK expression profile in st.293-R1{alpha} cells. The mRNA and protein of at least four GRK isoforms, namely GRK2, 3, 5, and 6 were found to be present in HEK 293 cells, as well as in human pregnant myometrial cells, as shown by RT-PCR using specific primers and immunoblot analysis using GRK isoform-specific antibodies (Fig. 7AGo). Previous studies failed to identify GRK3 expression in HEK 293 cells (30); however, the mRNA and protein of this GRK isoform was found to be present in our HEK 293 cells. To identify the specific isoforms of GRKs involved in the CRH-R1{alpha} homologous desensitization, we used specific antibodies against GRKs 2, 3, 5, and 6 to inhibit CRH-induced CRH-R1{alpha} desensitization. Treatment of SLO-permeabilized cells with anti-GRK3 and GRK6 antibodies almost inhibited the desensitization response by 90 ± 4% and 40 ± 5%, respectively. By contrast, anti-GRK2 and anti-GRK5 produced insignificant effects (6 ± 7% and 12 ± 5%, respectively, P > 0.05) (Fig. 7BGo). These results indicate that, in HEK 293 cells, GRK3 and GRK6 are the primary GRKs mediating the homologous CRH-R1{alpha} desensitization.



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  		Fig. 7. GRK Isoforms Involved in CRH-R1{alpha} Desensitization

A, Detection of GRKs mRNA (by RT-PCR) and protein (by Western blot) expression in human pregnant myometrial and HEK 293 cells. Lane M is the DNA ladder marker, lane NC is the negative control. Identical results were obtained from myometrial cells from 4 individual biopsies. B, Inhibition of GRK-mediated CRH-R1{alpha} desensitization in HEK 293 cells by GRK-isoform-specific antibodies. HEK 293 cells were permeabilized with SLO in the presence or absence of GRK2, 3, 5, and 6-specific antibodies (1:100) for 25 min to allow their access into the cytoplasm. The effect of GRK antibodies on the CRH-induced homologous desensitization (induced by pretreatment with 100 nM CRH for 30 min) was determined by measurement of the CRH-induced cAMP production. Data represent the mean ± SEM of two estimations from five independent experiments. *, P < 0.05 compared with cells without CRH pretreatment (nondesensitized); +, P < 0.05 compared with desensitized cells not receiving antibodies. Basal cAMP levels 1.3 ± 0.7 pmol/ml/2 x 105 cells.

 
Further experiments were performed to elucidate the pattern of GRK activation and membrane translocation in response to agonist-occupied receptor. Distribution of GRK immunoreactivity was monitored in cell membrane and cytosolic fractions prepared from st.293-R1{alpha} cells, treated with 100 nM CRH for various time intervals (0–15 min), to detect potential GRK translocation upon agonist stimulation. In resting st.293-R1{alpha} cells, GRKs 2 and 3 were mainly detected in the cytoplasm, whereas GRKs 5 and 6 immunoreactivity was primarily found in the cell membrane. CRH treatment for up to 15 min did not alter the protein expression levels of GRK2 and 5 in the cytosolic and membrane fractions. However, CRH induced significant changes in the subcellular distribution of GRK3 within 3 min, resulting in a 60% increase in the membrane fraction content of GRK3 (Fig. 8Go). The subcellular distribution of GRK6 was not altered after CRH treatment, a finding that is consistent with the view that GRK6 is constitutively localized in the cell membrane.



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  		Fig. 8. Time Course of GRK Subcellular Distribution after CRH-R1{alpha} Activation in st.293-R1{alpha} Cells

Left panels, Immunoblots of cytosolic and membrane fractions prepared from st.293-R1{alpha} cells, stimulated with or without CRH (100 nM) for different time intervals. Equal amounts of proteins (50 µg) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to immunoblotting with specific GRKs antibodies. Densitometry scanning was carried out to quantify agonist-induced specific GRK activation and translocation to the plasma membrane. Right panels, Mean ± SEM of three independent experiments. *, P < 0.05 compared with basal.

 
For desensitization of many GPCRs, Gß{gamma} subunits are required to target specific GRKs to the membrane-associated receptor substrates (31). To address this, we investigated the potential of GRK3 or GRK6 to form a binary complex with Gß{gamma}. Membranes from st.293-R1{alpha} cells, incubated in the presence or absence of CRH (100 nM), were solubilized and immunoprecipitated with GRK3 or six antibodies. The Gß{gamma}-protein subunits copurified with the GRKs, were detected by immunoblotting using a Gß common antibody that recognizes the Gß1-, Gß2-, Gß3-, or Gß5-subunits. Plasma membranes from native HEK 293 cells were used as controls for the blotting antisera and specific immunoreactivity was detected at 35–36 kDa with anti-Gß antibody (data not shown). Our results demonstrated that when GRK3 was precipitated under basal conditions small amounts of Gß-proteins were found coassociated; however, when the receptor was activated by CRH (100 nM),there was a significant 2- to 2.5-fold increase in the amount of Gß-proteins that could be coimmunoprecipitated with GRK3 (Fig. 9AGo). Association of the GRK-Gß{gamma} complex was apparent within 5 min of CRH treatment and was stable for at least 15 min of CRH treatment. Furthermore, addition of 1 µM Gß peptide during the immunoprecipitation completely blocked the CRH-induced increase in the amount of Gß-protein precipitated (data not shown). By contrast, under basal or CRH-stimulated conditions, Gß immunoreactivity was not associated with GRK6, consistent with previous reports (30). Resolved proteins from the immunoprecipitated membrane fractions were also immunoblotted with a GRK6 antibody to ensure equal amount of protein loading (Fig. 9AGo, inset).



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  		Fig. 9. Identification of CRH-induced GRK, Gß-Subunit and CRH-R1{alpha} Complex Formation by Coimmunoprecipitation in st.293-R1{alpha} Cells

Membrane fractions were prepared from st.293-R1{alpha} cells, stimulated with or without CRH (100 nM) for 15 min and were immunoprecipitated with specific (A) GRK3 or 6 antibodies, or (B) Gß-subunit antibodies, or (C) CRH-R1/2 antibodies. Proteins were resolved on SDS-PAGE gels, followed by immunoblotting with (A) Gß-subunit antibodies or (B and C) GRK3 or 6 antibodies to identify potential complex formation. Densitometry scanning was employed to quantify agonist-induced association between GRK, Gß-subunit, and CRH-R1{alpha}. Representative immunoblots are presented, and data represent the mean ± SEM of two estimations from three independent experiments. *, P < 0.05 compared with basal (CRH untreated) cells.

 
Identical results were observed when a reciprocal protocol was employed, in which the anti-Gß antisera was used for immunoprecipitation, and after protein separation by SDS-PAGE, coprecipitated GRK3 proteins were detected by GRK3 antibodies (Fig. 9BGo). Resolved proteins from the immunoprecipitated membrane fractions were also immunoblotted with a Gß-protein antibody to ensure equal amount of protein loading (Fig. 9BGo, inset).

To confirm the direct binding of GRK3 and GRK6 with the activated CRH-R1{alpha} receptor, aliquots of the same membrane fractions were immunoprecipitated with anti-CRH-R1 antibody, and the resolved proteins were immunoblotted with a GRK3 or GRK6 antibody. CRH treatment led to a 2.7-fold increase, compared with unstimulated membranes, in the amount of GRKs coprecipitated with the CRH-R1{alpha} receptor (Fig. 9CGo), suggesting that CRH enhanced formation of CRH-R1{alpha}/GRK3 and CRH-R1{alpha}/GRK6 complexes. Resolved proteins from the immunoprecipitated membrane fractions were also immunoblotted with anti-CRH-R1 antibody to confirm equal amount of CRH-R1 immunoprecipitation (Fig. 9CGo, inset).

Analysis of Potential Phosphorylation Sites for GRKs in the C Terminus of CRH-R1{alpha}
The distal part of the CRH-R1{alpha} carboxyl terminus contains a cluster of Ser/Thr residues (Ser396-Ser405) that represent potential targets of GRK phosphorylation action. To address this, we used site-directed mutagenesis to create CRH-R1{alpha} receptors, in which Ser396, Thr399, Ser400, or Ser405 were replaced by alanine (Fig. 10AGo). Wild-type and mutant CRH-R1a receptors [(S396A)CRH-R1{alpha}, (T399A)CRH-R1{alpha}, (S400A)CRH-R1{alpha},and (S405A)CRH-R1{alpha}], were transiently expressed in HEK 293 cells. These mutant receptors were expressed normally at the cell membrane (Fig. 10BGo) and were able to bind 125I-tyr°-CRH with binding affinity and maximum binding capacity that were similar to that of the wild-type CRH-R1{alpha} (Table 2Go). The mutant receptors (S396A)CRH-R1{alpha}, (S400A)CRH-R1{alpha}, and (S405A)CRH-R1{alpha}, exhibited homologous desensitization responses to CRH that were comparable with that of the wild-type receptor (Fig. 11AGo). However, replacement of Thr399 by alanine led to a 50–60% reduction in CRH-induced desensitization of the CRH-R1{alpha}, and a corresponding significant reduction in CRH-induced receptor phosphorylation compared with wild-type CRH-R1{alpha} receptors (Fig. 11BGo).



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  		Fig. 10. CRH-R1{alpha} C Terminus Mutant Receptor

A, Schematic representation of the Ser/Thr cluster present in the CRH-R1 distal portion of the C terminus. The amino acid residues in black background indicate single amino acids that were substituted to alanines using site-directed mutagenesis. B, Comparison by Western blot analysis of wild-type (w.t) and mutant CRH-R1{alpha} receptor expression in HEK 293 cells. Cell lysates from HEK 293 cells transiently expressing w.t or mutant CRH-R1 receptors were fractionated on SDS-PAGE, and subjected to immunoblotting with a specific CRH-R1/2 antibody as described in Materials and Methods. Identical results were obtained from three independent transfection experiments.

 

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  		Table 2. Expression and 125I-tyr-oCRH Binding Characteristics of Wild-Type and Mutant CRH-R1{alpha} Transiently Expressed in HEK 293 Cells

 


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  		Fig. 11. Role of CRH-R1 C-Terminus Ser/Thr Residues on Receptor Homologous Desensitization

A, Homologous desensitization characteristics of wild-type (w.t) and mutant CRH-R1{alpha} receptors. HEK 293 cells transiently expressing w.t and mutant CRH-R1{alpha} receptors were pretreated with or without CRH (100 nM for 30 min) to induce desensitization. After extensive washing, the cAMP response after a second 100 nM CRH stimulus for 15 min was determined. Data represent the mean ± SEM of two estimations from three independent experiments. *, P < 0.05 compared with cells without CRH pretreatment. +, P < 0.05 compared with desensitization response of cells expressing w.t CRH-R1{alpha}. Basal cAMP levels 1.9 ± 0.4 pmol/ml/2 x 105 cells. B, In vitro phosphorylation of w.t and (T399A)CRH-R1{alpha}. HEK 293 transiently expressing w.t and (T399A)CRH-R1{alpha}, were treated with 100 nM CRH for 30 min, in the presence of 32P. Phosphorylated CRH-Rs were solubilized, immunoprecipitated with specific CRH-R1/2 antibodies, fractionated on SDS-PAGE, and were subjected to autoradiography (–70 C, 10–14 d) using intensifying screens.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
During human pregnancy and labor, GPCR desensitization and down-regulation might regulate myometrial contractility, by altering the myometrial response to a variety of agonists (32, 33, 34, 35), thus limiting the effectiveness of tocolytic agents (35, 36). In this study, we provide evidence that the ability of the CRH-R to activate myometrial adenylyl cyclase, a signaling pathway associated with smooth muscle relaxation, is markedly reduced by homologous desensitization when exposed to high (10 nM or greater) concentrations of CRH. Although the concentrations of immunoreactive CRH present in maternal plasma during pregnancy are lower than those required to induce receptor desensitization, it is possible that levels of locally produced CRH are sufficiently high to achieve receptor desensitization. In addition, as the levels of CRH progressively increase during pregnancy, it is likely that desensitization of the CRH-R and reduced signaling efficiency provide an important mechanism for regulating CRH responsiveness in the myometrium during pregnancy and labor.

Because the human myometrium expressed multiple CRH-R subtypes and receptor isoforms (7), we created a model system in HEK 293 cells, to study homologous desensitization of the CRH-R1{alpha}, the principal CRH-R isoform-mediating CRH actions in human myometrium during pregnancy and is also widely expressed in the central nervous system and other peripheral tissues. Although the levels of CRH-R1{alpha} expression in HEK 293 cells are significantly greater (by 50- to 100-fold) than in human myometrium, and subtle differences might be present, our previous studies (18, 19) have confirmed the suitability of this system for studying myometrial CRH-R1{alpha} signaling properties. Our studies confirmed that the CRH-R1{alpha} undergoes homologous desensitization to CRH, leading to significant reduction in CRH-R1{alpha}-Gs-protein coupling and a corresponding decrease in generation of cAMP, the primary intracellular mediator of CRH effects in most tissues (3). In contrast with other members of the class B GPCR superfamily (37), desensitization of the CRH-R1{alpha} is a relatively slow process, with a half-maximal desensitization occurring approximately 40 min after treatment, and full recovery of CRH-R1{alpha} responsiveness not apparent until 2 h. The kinetics of homologous desensitization of CRH-R1{alpha} in our experimental model were quite distinct from other models used previously to investigate this phenomenon, suggesting that cell-specific mechanisms may be involved. For example, in the IMR-32 human neuroblastoma cell line, maximal desensitization of native CRH-R1 receptor was observed within 30 min with only partial recovery (38), whereas in mouse fibroblasts overexpressing CRH-R1, desensitization was evident minutes after CRH treatment with maximal reduction within 1 h (26). In human retinoblastoma Y-79 cells, which express CRH-R1, the time-dependent reduction in CRH-stimulated intracellular cAMP accumulation had a t1/2 of 38 min and was slowly reversible with a full restoration of cAMP responsiveness to CRH 24 h after removal of CRH desensitization stimulus (25).

For many but not all GPCRs, the homologous desensitization process involves receptor phosphorylation that subsequently increases interaction of the receptor with members of the arrestin family of proteins (21, 39). The binary complex formed between the phosphorylated GPCR and arrestin initiates two important intracellular processes, receptor uncoupling and internalization, that are involved in both the desensitization and the recovery of cellular responses. Our current study indicates that PKA is not involved in homologous desensitization of the CRH-R1{alpha}, in agreement with previous data (24), although PKA-induced receptor phosphorylation can alter the functional activity of the CRH-R1{alpha} (19). Other studies have shown that PKC is important only for heterologous desensitization of CRH-R (40, 41).

To analyze the role of GRKs in mediating CRH-R1{alpha} phosphorylation and homologous desensitization, we relied upon the endogenous GRK isoforms in HEK 293 cells stably expressing the CRH-R1{alpha}. Although overexpression of GRK isoforms is a complementary approach, such an approach may prevent promiscuous interactions that occur normally. We found that HEK 293 cells express GRK2, 3, 5, and 6; initial experiments employing heparin or Zn2+, two widely used general inhibitors of GRK (42, 43), confirmed that GRKs participate in the process of CRH-R1{alpha} phosphorylation and homologous desensitization. Internalization experiments using confocal microscopy provided novel evidence that CRH-R1{alpha} desensitization is followed by receptor internalization and endocytosis in a dose- and time-dependent manner.

In our HEK 293 cellular system, GRK3, but not GRK2, appeared to be the major cytosolic endogenous GRK isoform involved in CRH-R1{alpha} homologous desensitization and after its activation, it required Gß{gamma}-subunits for its recruitment to the plasma membrane and association with the CRH-R1{alpha}. This selectivity is likely due to differences between GRK2 and GRK3 in the 28-amino acid domain in the C terminus that specifies interaction with the unique Gß{gamma}-subunits, which enable heterotrimeric G proteins to distinguish between different GPCRs (31). By contrast, GRK6, the main membrane-bound GRK isoform mediating CRH-R1{alpha} homologous desensitization, was able to interact with the CRH-R1{alpha} in a Gß-subunit-independent manner, consistent with the view that it is constitutively associated with the plasma membrane via a covalently attached palmitate (44). Physiological relevance derives from the observation that myometrial GRK6 is up-regulated at term in human pregnancy (45) and mediates myometrial ß-adrenergic receptor desensitization (46), suggesting potential significance for GRK6 actions at the onset of labor.

Although preliminary, our results suggest that there are no temporal differences in GRK association and complex formation with the CRH-R1{alpha} in HEK 293 cells. After receptor activation, both GRK3 and GRK6 isoforms were found to associate with the CRH-R1{alpha} during a 15-min CRH treatment that corresponded to receptor desensitization. It is possible, however, that individual GRKs may target different domains or even residues of the CRH-R1{alpha}, and the subsequent distinct phosphorylation profile may have important functional consequences for the receptor. Similar interactions have been observed for other GPCRs (30).

Putative sites of GRK-mediated phosphorylation of GPCRs are difficult to predict because no obvious consensus sequence for receptor recognition and phosphorylation by individual GRKs has been identified. For many GPCRs, a diacidic motif upstream of the phosphorylated serine and threonine residues has been suggested as a potential consensus sequence (47, 48), although no such amino acid motifs are present in the CRH-R1{alpha} sequence. In addition, several studies have proposed Ser/Thr residues that are located in the distal portion of the cytoplasmic tail of many GPCRs, including the ß2-adrenergic, the type 1A angiotensin II (AT1A-R) and V2 vasopressin receptor (49, 50, 51), as potentially important in receptor desensitization. The distal portion cytoplasmic tail of the CRH-R1{alpha} contains many Ser/Thr residues (eight of the last 20 amino acids), suggesting that one or more of these residues may be sites for phosphorylation. Therefore, we generated a series of mutant CRH-R1{alpha} receptors (Ser396-Ser405) in which a Ser/Thr was replaced with Ala. The expression and signaling characteristics of the mutant CRH-R1{alpha} receptors were similar to wild type. However, one specific replacement, Thr399->Ala reduced agonist-induced CRH-R1{alpha} phosphorylation as well as desensitization. This is important because receptor phosphorylation is not an absolute requirement for desensitization. In contrast, Ser396, Ser400, or Ser405 did not appear to participate in receptor desensitization. Further studies will address whether Thr399 or any of the other serines in the distal portion of the C terminus are also important for ß-arrestin binding and internalization of the CRH-R1{alpha} receptor. Because alanine substitution of Thr399 did not completely prevent CRH-induced phosphorylation or desensitization of the CRH-R1{alpha} receptor, it is likely that additional amino acid residues serve as putative GRK phosphorylation sites

In conclusion, CRH binding to CRH-R1{alpha} activates molecular mechanisms that, in addition to specific G protein and second messenger stimulation, lead to receptor desensitization and internalization and involve multiple GRK isoforms, Gß {gamma}-subunits and ß-arrestin (Fig. 12Go). Specific amino acids located in the C terminus of the CRH-R1{alpha} appear to be important potential phosphor-acceptor sites for GRK-dependent phosphorylation and desensitization demonstrating the importance of the carboxyl tail for regulation of receptor signaling potency. This process might also initiate a second wave of distinct signaling events and indeed our preliminary data suggest that CRH-R1{alpha} internalization is important for ERK signaling (our unpublished observations).



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  		Fig. 12. Schematic Representation of the Proposed Mechanism CRH-R1{alpha} Homologous Desensitization in HEK 293 Cells

According to this model, agonist-induced receptor activation leading to stimulation of the Gs-AC pathway, also induces GRK3 and 6 recruitment and complex formation with the CRH-R1{alpha} receptor. GRK3 translocation to the plasma membrane also requires interaction with Gß{gamma} subunits. This is followed by receptor phosphorylation, and homologous desensitization via a ß-arrestin-dependent mechanism. The amino acid residue Thr399 of the CRH-R1{alpha} C-tail appears to be one of the target residues important for GRK actions. Subsequently, agonist-induced CRH-R1{alpha} activation initiates receptor endocytocis and internalization.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Chemicals
Radioiodinated ovine CRH and human/rat CRH, were obtained from Penninsula Laboratories (Merseyside, UK). The mammalian expression vector pcDNA3.1(–) was obtained from Invitrogen Life Technologies (Paisley, Scotland, UK). Dithiothreitol, GDP, forskolin, 2-[N-morpholino]ethane sulfonic acid, 1,4-dioxane, triethylamine, and all other chemicals were purchased from Sigma (Poole, Dorset, UK). Waters Sep-Pak C18 columns were obtained from Millipore (UK) Ltd. (Watford, Hertfordshire, UK). Polyclonal G protein rabbit antibodies and the cAMP assay kits were obtained from Dupont-NEN (Hertfordshire, UK). GRK antibodies were obtained from Santa Cruz Biotechnology; (Santa Cruz, CA). SLO and all other chemicals were purchased from Sigma (Poole, Dorset, UK). Protein-A Sepharose beads (CL-4B) was purchased from Pharmacia (Uppsala, Sweden). [{alpha}-32P]-GTP and reagents for enhanced chemiluminescence were obtained from Amersham International (Little Chalfont, Buckinghamshire, UK). 4-Azidoanilide-HCl, 1-(3-dimethylamino propyl)-3-ethylenecarbodiimide hydrochloride were purchased from Sigma Aldrich (Dorset, UK). Synthetic oligonucleotide probes, PCR and cloning reagents, culture media and enzymes were purchased from Invitrogen Life Technologies. All other chemicals were purchased from BDH (VWR International, Merck House, Poole, Dorset, OK).

Subjects and Culture of Myocytes
Pregnant myometrial biopsies (n = 4) were obtained from women undergoing elective cesarean section at term before the onset of labor for nonmaternal problems. The biopsy site was standardized to the upper margin of the lower segment of the uterus in the midline. This provides the closest approximation to the upper segment of the uterus. These studies were approved by the local ethical committee and informed consent was obtained from all patients.

The tissue was immediately placed in 20 ml of ice-cold DMEM culture medium containing antibiotics (200 IU penicillin/ml and 200 mg streptomycin/ml). Myocytes were prepared by enzymatic dispersion as previously described (19). The cells were cultured at 37 C in a humidified atmosphere of 95% air and 5% CO2 until confluent.

Site-Directed Mutagenesis and Transfection of Wild-Type/Mutant CRH-R1{alpha} to HEK 293 Cells
Human CRH-R1{alpha} cDNA was subcloned into the mammalian cell expression vector pcDNA3.1(–) (Invitrogen Life Technologies) and was used as a template for mutagenesis using the PCR overlap extension method (52) with Pfu polymerase (Stratagene, La Jolla, CA). The entire regions amplified by PCR were sequenced to ensure the fidelity of the mutant cDNAs and confirm presence of mutations. DNA sequence analysis was performed by the Core facility of the Department of Biological Sciences, University of Warwick.

Human wild-type or mutant CRH-R1{alpha} cDNAs were transfected in HEK 293 cells using Lipofectamine reagent (Invitrogen Life Technologies) as previously described (19). Cells were grown in medium tissue culture flasks in a culture medium consisting of high-glucose DMEM (Invitrogen Life Technologies) containing Glutamax, 25 mM HEPES, 10% FCS, 1% penicillin/streptomycin. For generation of cell lines stably expressing CRH-R1{alpha} (st.293-R1{alpha}), transfected cells were grown in DMEM in the presence of G418 (500 µg/ml) to select for transfected cells and those surviving were subcultured and maintained in DMEM containing G418 (100 µg/ml). In some experiments, st.293-R1{alpha} cells were transiently transfected with the dominant-negative arrestin, [arr-2 (319–418)] cDNA subcloned in pcDNA3 (Invitrogen Life Technologies).

Total RNA Extraction and PCR
Total RNA was prepared from HEK 293 or myometrial cells using RNeasyTM Total RNA Kit (QIAGEN, Crawley, UK) according to the manufacturer’s guidelines. First-strand cDNA synthesis was performed using ribonuclease reverse transcriptase (Invitrogen Life Technologies).

All PCRs were carried out using Taq DNA Polymerase (Invitrogen Life Technologies) with 200 ng of cDNA for each amplification. The set of primers used for the amplification of GRKs 2, 3, 5, and 6 are listed in Table 3Go. cDNAs were amplified at 95 C (1 min); 57 C (1 min); 72 C (1 min); in a total of 30 cycles with a final extension step at 72 C for 7 min. Ten microliters of the reaction mixture were subsequently electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide, using a 1-kb DNA ladder (Invitrogen Life Technologies) to estimate the band sizes. As a negative control for all of the reactions, distilled water was used in place of the cDNA. The resultant PCR products were sequenced in an automated DNA sequencer and the sequence data was analyzed using Blast Nucleic Acid Database Searches from the National Centre for Biotechnology Information.


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  		Table 3. Oligonucleotide Primers Used for the Amplification of GRKs 2, 3, 5, and 6 mRNA from HEK293 and Human Pregnant Myometrial Cells

 
Permeabilization of st.293-R1{alpha} Cells
SLO was used to permeabilize st.293-R1{alpha} cells. Cells grown in 24-well culture plates until 60–70% confluency were briefly washed with PBS, followed by addition of 0.5 ml HBSS culture media containing 30 mM HEPES without Ca2+, for 15 min at 37 C. Ten micrograms/milliliters SLO were added for 25 min and trypan blue staining was used to identify permeabilized cells. The agent for delivered to the cytosol was included during this step (heparin 1 µM, 1 min or 1:100 GRK Abs, 25 min). Cell membrane resealing was induced by the addition of 1.5 ml of ice-cold HBSS media containing 30 mM HEPES and 2 mM CaCl2. Over 80% of the permeabilized cells would reseal within 60 min incubation at 4 C, evident by the reduction in number of cells positively stained by trypan blue. SLO-permeabilized st.293-R1{alpha} cells were incubated for 25 min with various antibodies specifically recognizing the C terminus of GRKs 2, 3, 5, 6 (1:100), before pretreatment with 100 nM CRH for 30 min, and measurement of cAMP accumulation induced by a second stimulation with 100 nM CRH for 15 min.

Immunoprecipitation and Western Blot Analysis Studies
HEK 293 cells were washed with 10 ml PBS, detached from the flask and collected by centrifugation at 1000 rpm for 10 min. The pellet was resuspended in 1.5 ml of Dulbecco’s PBS homogenization buffer containing 2 mM EDTA, 10 mM MgCl2, 0.15% BSA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and homogenized using a glass homogenizer. The homogenate was centrifuged at 2000 rpm for 30 min at 4 C and the supernatant was collected and centrifuged at 19,000 rpm for 1 h at 4 C. The resulting supernatant (cytosolic fraction) was separated and stored, whereas the pellet (membrane fraction) was resuspended in 5 ml of homogenization buffer and recentrifuged as before for 1 h. The final pellet was resuspended in 1 ml of homogenization buffer, aliquoted, and stored at –70 C.

For Western analysis experiments, samples (cytosolic and membrane fractions) were resuspended in Laemmli’s buffer (30 µl) (42) and boiled for 5 min. Samples were then subjected to gel electrophoresis using discontinuous SDS-PAGE slab gels (10% running, 5% stacking). Molecular weight markers dissolved in solubilization buffer were also electrophoresed.

For immunoprecipitation experiments, membrane fractions (200–400 µg/50 µl) of st.293-R1{alpha} cell were precipitated by centrifugation (5 min at 13,000 rpm) and solubilized in 60 µl of 2% sodium dodecyl sulfate while kept in ice. Insoluble material was removed by centrifugation, followed by addition of 100 µl of buffer containing 1 mg/ml aprotinin, 1 mg/ml leupeptin, 100 nM PMSF, 100 nM sodium orthovanadate, 5% total BSA) to the supernatant. Antiserum for GRK3 or GRK6 (4 µg/ml) or CRH-R1/2 (25 µg/ml) or 10 µl of anti-Gßcommon (Calbiochem, San Diego, CA) was added and samples were rotated for 4 h at 4 C. Then, 50 µl of protein A-Sepharose 4B beads were added and the incubation was continued at 4 C overnight The beads were collected by centrifugation (5 min at 13,000 rpm) and washed three times in 100 mM Tris-HCl buffer (pH 7.4) containing 1 mg/ml aprotinin, 1 mg/ml leupeptin, 100 nM PMSF, 100 nM sodium orthovanadate, 1% total BSA. After centrifugation, the pellet was resuspended in 50 µl Laemmli’s buffer, boiled for 5 min and resolved by SDS-PAGE.

The resolved proteins were then transferred onto nitrocellulose membranes using a semidry electroblotter (Whatman, Biometra, Goettingen, Germany) at 100 mA for 45 min. To minimize nonspecific binding of antibody to the membrane matrix, nitrocellulose membranes were incubated for 2 h at room temperature with gentle agitation, with blocking solution (1–5% low-fat dried milk powder, 0.05–0.1% Tween 20, in PBS). After three washes with PBS-0.1% Tween, 10 min each, the membranes were incubated with primary anti-GRK antibodies (affinity purified rabbit polyclonal antibodies raised against carboxy terminus of each GRK; Santa Cruz Biotechnonolgy, Inc). Primary antisera were used at 1:1000 for GRK2, 1:500 for GRK3, 1: 500 for GRK5 and 1: 500 for GRK6 dilution in PBS-0.1% Tween for 1 h at room temperature on an orbital shaker. The membranes were then washed six times in 20 ml PBS-0.1% Tween, with gentle agitation for 10 min, followed by incubation with the secondary horseradish peroxidase-conjugated antirabbit Ig (Santa Cruz Biotechnonolgy, Inc.) (1:2500 dilution in PBS-0.1% Tween) for 1 h at room temperature followed by further six washings for 10 min each, with PBS-0.1% Tween. Antibody complexes were detected using an enhanced chemiluminescence detection kit (Amersham International) according to the manufacturer’s instructions.

CRH-R Radioreceptor Assay
Radioiodinated ovine CRH binding characteristics in st.293-R1{alpha} or HEK 293 cells transiently expressing wild type or mutant CRH-R1{alpha} receptors were determined as previously described (19). The binding data were analyzed using the EBDA program (53) and ligand (54).

Receptor Desensitization/cAMP Studies
Primary myometrial cells or HEK 293 cells transiently expressing wild type or mutant CRH-R1{alpha} were plated in 24-well dishes up to 80% confluency and pretreated with various concentrations of human/rat CRH (1–100 nM) in plain DMEM for various time periods. At the end of the incubation period, the medium was removed and the cells were rinsed with plain medium, and then stimulated with 100 nM CRH for 15 min at 37 C. The reaction was terminated by addition of 0.3 M HCl and cAMP production was determined by RIA as previously described (19). In some experiments, after CRH pretreatment, cells were allowed to rest in fresh plain DMEM for various time intervals (0–2 h) before stimulation with 100 nM CRH for 15 min at 37 C.

Receptor Phosphorylation Assay
In vitro phosphorylation assays in HEK 293 cells expressing wild-type or mutant CRH-R1{alpha} were carried out as previously described (19).

GTP-AA Photolabeling and Immunoprecipitation of G{alpha} Subunits
Agonist-induced receptor-Gs protein interaction was determined by using [{alpha}-P32]-GTP-AA following a method described previously (19).

Receptor Immunofluorescence and Internalization Studies
HEK 293-R1{alpha} cells, seeded on glass coverslips pretreated with 3-(aminopropyl)triethoxy silane, were grown in six-well plates until 70–80% confluency. After treatment with CRH (0–100 nM) for various time intervals (15–60 min), cells were fixed with 4% paraformaldehyde in PBS. To reduce nonspecific binding, cells were incubated with 3% BSA in PBS-Triton X-100 (0.01%) for 1 h at room temperature, followed by a washing step [three times for 5 min with PBS-Triton X-100 (0.01%)]. The incubation with the goat polyclonal anti-CRH-R1/2 antiserum (1:100) (Santa Cruz Biotechnology, Inc.) was carried out at 4 C overnight. Cells were washed with PBS-Triton X-100 as before, and incubated with donkey antigoat Alexa-Fluor 594 antibody (1:400 in PBS-Triton X-100) (Molecular Probes, Cambridge, UK) for 1 h at room temperature. Coverslips were washed with PBS and mounted on microscope slides with 50% glycerol in PBS. Cells were examined under an oil immersion objective (x63) using a Leica model DMRE laser scanning confocal microscope (Leica Microsystems, UK, Buckinghamshire, Milton, Keynes, UK) with TCS SP2 scan head. Laser 543 nm at 50% of power and a filter selective for Texas Red fluorescence (570 nm) were used. Optical sections (0.5 µm) were taken, and representative sections corresponding to the middle of the cells were presented. After indirect immunofluorescent staining, no specific fluorescence was observed in untransfected HEK 293 cells or in st.293-R1{alpha} cells treated with secondary antibody Alexa-Fluor 594 only (data not shown). The images were manipulated with Leica (5x zoom) and Adobe Photoshop softwares.

Statistical Analysis
Data are shown as the mean ± SEM of each measurement. Comparison between group means was performed by ANOVA, and P < 0.05 was considered significant. The relative density of the bands was measured by OD scanning using the software Scion Image-Beta 3b for Windows (Scion Corp., Frederick, MD).


   ACKNOWLEDGMENTS
 
We would like to thank Dr. J. Benovic (Thomas Jefferson University Medical School, Philadelphia, PA) who kindly provided us with cDNA for ß-arrestin (319–418) dominant-negative mutant, and Dr. Andrew Blanks (University of Warwick) for expert guidance and advice on the confocal microscopy experiments. We would also, like to acknowledge Dr. H. Randeva and Dr. E. Karteris for useful discussions.


   FOOTNOTES
 
This work was supported by a Wellcome Trust Career Development Award (to D.G.) and a Coventry General Charities award (to E.W.H.). Part of this work was presented at the 86th Annual Meeting of The Endocrine Society, New Orleans, Louisiana, June 16–19, 2004, and was supported by an Endocrine Society Travel Award.

First Published Online October 21, 2004

Abbreviations: AC, Adenylyl cyclase; CRH-R1, CRH receptor type 1; GPCR, G protein-coupled receptors; GRK, GPCR kinase; HEK, human embryonic kidney; PKA, protein kinase A; PKC, protein kinase C; PMSF, phenylmethylsulfonyl fluoride; SLO, streptolysin O; UCN, urocortin.

Received for publication July 7, 2004. Accepted for publication October 15, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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