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The Kidney Institute (M.P., L.D.Q.), Department of Internal Medicine, University of Kansas Medical Center, Kansas City, Kansas 66160; NORAK Biosciences (R.H.O., R.D.C.), Morrisville, North Carolina 27709; Division of Endocrinology (D.G.-P., R.F.S.), Department of Medicine, Duke University Medical Center, Durham, North Carolina 27701; and Division of Endocrinology (L.M.L.), Diabetes and Medical Genetics, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425
Address all correspondence and requests for reprints to: L. Darryl Quarles, M.D., Summerfield Endowed Professor of Nephrology, University of Kansas Medical Center, MS 3018, 3901 Rainbow Boulevard, 6018 Wahl Hall East, Kansas City, Kansas 66160. E-mail: dquarles{at}kumc.edu.
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q signaling. In addition, we observed lower circulating PTH levels and an attenuated increase in serum PTH after hypocalcemic stimulation in ß-arrestin2 null mice, suggesting a functional role of ß-arrestin2-dependent desensitization pathways in regulating CASR function in vivo. We conclude that GRKs and ß-arrestins play key roles in regulating CASR responsiveness in parathyroid glands. |
INTRODUCTION |
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i- and Gq-protein-coupled receptor (GPCR) that mediates PTH secretion by the parathyroid glands in response to changes in extracellular calcium concentrations (1). Calcium-sensing receptor (CASR) belongs to the class 3 family of GPCRs that include mGlu receptors, 
-aminobutyric acid (GABA)B receptors, and many olfactory, pheromone, and taste receptors (www.GPCR.org). CASR is continuously exposed to extracellular calcium and yet remains exquisitely sensitive to small changes in serum calcium. Consequently, ligand-mediated homologous desensitization may be an important control point for regulating CASR signaling. Homologous desensitization of GPCRs typically involves G protein-coupled kinases (GRKs) and ß-arrestins (2, 3, 4). GRKs, a seven-member family of serine/threonine kinases (GRK1–7), phosphorylate clusters of serine and threonine residues in the C termini of agonist-stimulated GPCRs, which permits binding of members of the ß-arrestin family (3, 4, 5, 6, 7, 8). The arrestin family, which consists of four isoforms, two expressed only in the visual system (S-antigen and C-arrestin) and two that are ubiquitously expressed (ß-arrestin1 and ß-arrestin2), bind to phosphorylated C termini of GPCRs, resulting in both an uncoupling of the receptor from its cognate G proteins and targeting of the receptor for internalization (9). Specific GPCRs differ in their capacity to form stable receptor-ß-arrestin complexes and traffic into endocytic vesicles (10).
There is limited information regarding the mechanisms of homologous desensitization of the class 3 family of receptors. These receptors represent a unique subclass of GPCRs that bear little sequence or structural homology to other GPCRs, except for the presence of the seven transmembrane-spanning domain topology (11, 12, 13, 14, 15, 16, 17, 18). Both ß-arrestin1 and ß-arrestin2 have been implicated in mGluR1 desensitization and internalization (12, 14), but the effects of GRKs appear to be receptor specific. For example, GRK2 and GRK3, but not GRK4, have been reported to regulate mGluR5 (18), whereas GRK5 has been shown to desensitize mGluR1a (12). In contrast, GRK4 appears to play a major role in the regulation of mGlu1 and GABA receptor desensitization (14, 15). In addition, desensitization of mGluR and GABAB receptors also can occur through mechanisms not involving ligand-induced receptor phosphorylation (13, 19, 20).
To date, the roles of GRKs and ß-arrestins in the regulation of CASR function have not been reported. In the present study, we investigated the desensitization, phosphorylation, and internalization of CASR by GRKs and ß-arrestins in cell cultures and in ß-arrestin2 null mice.
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, upper panel). We also determined the expression of the seven known GRK isoforms in human parathyroid glands by RT-PCR using GRK1–7 specific primers (Fig. 1A). We detected the widely expressed cytosolic GRK2 and GRK3 as well as the ubiquitous membrane-associated GRK5 and GRK6, but were unable to detect GRK1 and GRK7 transcripts in human parathyroid glands under the conditions studied (Fig. 1A). We also detected GRK4 transcripts (confirmed by sequencing) in the parathyroid gland, which was unanticipated because prior studies had identified limited expression of GRK4 (21). Because we measured GRK expression at the tissue level, we cannot be certain that GRK4 is expressed in PTH-secreting cells, however functional data indicate that GRK4 can regulate the function of CASR (Fig. 1B), which is limited to PTH-secreting cells in the parathyroid gland. As a control for these studies, we examined expression of ß-arrestin1 and ß-arrestin2 and GRK1–7 in human kidney cortex (Fig. 1A, lower panel). The kidney cortex also lacks GRK1 and GRK7 as well as GRK4. The expression in the parathyroid glands of GRK4 suggests a possible role of this GRK isoform in regulating CASR function (see below).
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). SRE-luciferase activity has been used as a measure of CASR activation in cells cotransfected with CASR and this promoter-reporter construct. ß-Arrestin1 and 2 as well as GRK2 and GRK4 each resulted in approximately 20–30% reduction in calcium-stimulated CASR activity. Cotransfection of ß-arrestins with GRK2 or GRK4 had additive effects to inhibit CASR function, resulting in an approximately 60% reduction in cal-cium-stimulated luciferase activity. GRK5 had lesser effects to inhibit agonist-stimulated CASR activation in both the absence and presence of cotransfected ß-arrestins. GRK3 had similar effects to GRK2 (data not shown). We did not evaluate the effects of GRK6.
It is also possible that interactions between GRK2 and Gq might explain some of the inhibition of CASR signaling. To address this, we cotransfected GRK2 and the constitutively active Gq into HEK-293 cells expressing the SRE-luciferase reporter construct. We observed that GRK2 had a significant effect to inhibit SRE activation induced by the transfection of a constitutively active Gq QL indicating the presence of postreceptor actions of this GRK, whereas GRK5 did not block the actions of Gq QL (data not shown).
Defective PTH Secretion in ß-Arrestin2-Deficient Mice
To determine whether ß-arrestins are important for regulating signals through CASR in the parathyroid gland in vivo, we measured circulating PTH levels in serum from ß-arrestin2-deficient and sex-matched littermate control mice under basal conditions (Fig. 2A) and after stimulation of PTH secretion by EGTA-induced hypocalcemia (Fig. 2B). Based on our current understanding of arrestin-mediated down-regulation of GPCR activity, the absence of ß-arrestin2 might enhance activity of CASR leading to suppression of basal PTH secretion and an attenuated response to hypocalcemia. Consistent with this prediction, PTH levels were significantly lower in ß-arrestin2-deficient compared with control mice at similar serum calcium levels (Fig. 2A). Moreover, increments in serum PTH induced by hypocalcemia were significantly less in ß-arrestin2-deficient mice compared with wild-type mice, despite identical reductions in serum ionized calcium (Fig. 2B). Thus, ß-arrestin2 null mice appear to have a leftward shift in the calcium-PTH relationship.
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). To determine whether CASR undergoes agonist-stimulated phosphorylation, an amino-terminal FLAG epitope-tagged CASR construct was transiently transfected into HEK-293 cells and stimulated with extracellular calcium. In the absence of calcium, the FLAG-CASR was not phosphorylated (Fig. 3B), and in the presence of 10 mM calcium we detected phosphorylated CASR with a molecular mass of approximately 120 kDa. Both GRK2 and GRK4 enhanced basal FLAG-CASR phosphorylation and further enhanced calcium-mediated phosphorylation of FLAG-CASR (Fig. 3B).
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). Using the mammalian two-hybrid assays in which a GAL4/CASR cDNA was transiently transfected with VP16/ß-arrestin1 or 2 cDNA into COS-7 cells, we found that both ß-arrestins interacted with the C terminus of CASR (Fig. 4, B and C). Indeed, transfection of the CASR (877–1079) corresponding to the entire C terminus resulted in significant stimulation of the ß-gal reporter in the presence of cotransfected ß-arrestin1 or 2 (Fig. 4B). In the controls, we observed that no transactivation of the ß-gal reporter was found in cells transfected with the empty vector or VP16/ß-arrestins. In addition, transfection of CASR (636–805), corresponding to the region containing the intracellular loops, did not activate the reporter in the presence or absence of cotransfected ß-arrestin1 and 2. To determine whether GRK enhances ß-arrestin binding to the C terminus of CASR, we coexpressed GRK2, VP16/ß-arrestin1, and GAL4/CASR (877–1079) in COS-7 cells. CASR interaction with ß-arrestin1 was slightly enhanced by GRK2 (Fig. 4C). We did not assess whether GRK4 enhanced the interactions of ß-arrestin binding to the C terminus of CASR using the mammalian two-hybrid assay, but we examined this in translocation experiments (see below).
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). ß-Arrestin2-GFP distributed uniformly in the cytoplasm of mock-transfected cells (data not shown) and in control cells transfected with CASR without stimulation with calcium (Fig. 5A, left panel). ß-Arrestin-GFP translocates to the CASR in U2OS cells after treatment with 5 mM CaCl2 (Fig. 5A, right panel), as evidenced by the coalescence of CASR and ß-arrestin-GFP2 in small punctae along the plasma membrane in calcium-treated cells. Coexpression of GRK4 (but not GRK2–6, data not shown) enhanced the translocation of CASR, as evidenced by a greater redistribution of the cytoplasmic ß-arrestin2-GFP into punctae at the plasma membrane (Fig. 5B, compare left and right panels). These results, taken together with the phosphorylation studies, indicate that CASR is preferentially desensitized via GRK4 and ß-arrestins.
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). However, agonist-mediated internalization of CASR was not enhanced by cotransfection of GRK2 and/or ß-arrestin2.
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DISCUSSION |
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), but also displayed an attenuated increase in PTH in response to hypocalcemic stimulation (Fig. 2B). This apparent leftward shift in the calcium-PTH relationship is indicative of an increased sensitivity to calcium that is an expected response if ß-arrestin2 has a functional role to regulate CASR desensitization.
In vitro studies also support the importance of GRKs and ß-arrestins in regulating CASR desensitization (Figs. 1 and 3–5). In this regard, we found that CASR expressed in HEK-293 cells is phosphorylated, likely at clusters of serine/threonine residues present in its C terminus (Fig. 3A), in an agonist-dependent fashion, and that GRK2 or GRK4 enhanced the phosphorylation (Fig. 3B). We also demonstrated that GRKs and ß-arrestins had independent effects to inhibit CASR activity in vitro (Fig. 1B), similar to their roles in the homologous desensitization of other heptahelical receptors. The effects of GRKs and ß-arrestins were additive in their abilities to inhibit agonist-dependent CASR activation as assessed by functional assays (Fig. 1). In addition, we observed that ß-arrestin interacts with CASR by coimmunoprecipitation studies (Fig. 4A) and established that the C terminus of CASR is the site of ß-arrestin binding using mammalian-two-hybrid studies (Fig. 4B). These studies also showed the ability of GRKs to enhance ß-arrestin interactions with CASR (Fig. 4C). Finally, we demonstrated agonist-dependent translocation of a ß-arrestin-GFP fusion protein to CASR expressed in the plasma membrane of U2OS cells (Fig. 5A). Thus, the classical GRK-mediated receptor phosphorylation and binding of ß-arrestin to the phosphorylated receptor is responsible, at least in part, for homologous desensitization CASR in the parathyroid glands.
We also found that extracellular calcium stimulates the internalization of CASR (Fig. 6). The magnitude of CASR internalization that we observed was less than the 57% decrease in surface expression of CASR after stimulation with 10 mM Ca+2 reported by Gama et al. (22) using an ELISA in HEK-293 cells transiently expressing an M2-tagged human CASR-GFP fusion protein. Potential differences in methods, including our use of an M2-tagged rat CASR lacking GFP, lower calcium concentrations, and a different method for assessing receptor internalization, may explain the differences in the magnitude of receptor internalization. Overall, CASR exhibits a pattern of receptor-ß-arrestin interaction, as described by Oakley et al. (10), characterized by dissociation of the receptor-ß-arrestin complex at or near the plasma membrane and rapid recycling. Under the conditions studied, we were unable to demonstrate major differences in binding affinity of ß-arrestin2 compared with ß-arrestin1 (Fig. 4, B and C) that typically characterize rapid recycling receptors. This might be due to limitations of the mammalian two-hybrid assay using a portion of the receptor. It is possible that the difference could be present in intact cells using the full-length receptor.
Differences in the effects of various GRKs were observed on CASR activity. For example, GRK5 had less inhibitory activity compared with GRK2 and GRK4 (Fig. 1B). The presence of GRK4 in the parathyroid gland (Fig. 1) and its regulation of CASR function are novel findings. Previous studies suggested that GRK4 expression is abundant in the testis and limited in other tissues such as brain and kidney medulla (5, 6, 7). We show for the first time that GRK4 mRNA is expressed in parathyroid glands (Fig. 1A). We also demonstrated that GRK4 has a classical role in the desensitization of CASR in HEK-293 cells through mechanisms involving translocation of ß-arrestin2 to CASR (Fig. 5). Because GRK4 also plays a role in the desensitization of both mGluRs and GABA receptors (14, 15), and because GRK4 may promote the more rapid internalization of GPCR (15), it may be of biological importance in maintaining CASR responsiveness in the parathyroid gland as well as the function of other class 3 receptors. The importance of GRK4 might also be derived from its regulation by other factors, such as calmodulin, which does not regulate GRK2 (23).
In contrast, GRK2 did not enhance the recruitment of ß-arrestin to the plasma membranes in an agonist-dependent fashion, but directly inhibited Gq-dependent signaling in the absence of CASR. The ability of GRK2 to inhibit Gq-mediated SRE activation in the absence of CASR is consistent with the ability of GRK2 to inhibit signaling by binding to Gq via its amino-terminal regulators of G protein signaling (RGS) homology (RH) domain or to ß-subunits via its C-terminal domain (15, 19, 24, 25, 26). Indeed, RGS-like domains are present in GRK2 and GRK3 but absent in GRK4, GRK5, and GRK6 (15, 24). GRK2, but not GRK4, also contains a pleckstrin homology consensus caveolin binding motif, and GRK2 interactions with caveolin can inhibit GRK2-dependent phosphorylation of GPCRs (27). A differential role of GRK4 relative to GRK2 has been reported for homologous desensitization of D1 receptors in renal proximal tubule cells (28) and muscarinic acetylcholine receptors (29). Additional studies in GRK2 and GRK4 null mice will be needed to establish the physiological importance of GRK2- and GRK4-dependent regulation of CASR function in the parathyroid gland.
CASR may also interact with other proteins that modulate its recycling. Prior studies indicate that CASR likely translocates to caveolae where it binds to caveolin (30), findings consistent with our confocal studies showing localization of CASR and ß-arrestin in membrane pits (Fig. 5). In addition, we have shown that the C terminus of CASR binds to filamin (31), a cytoskeletal protein that appears to enhance the internalization and degradation of the calcitonin receptor (32). Members of the Homer family bind to metabotropic glutamate receptor 1, leading to greater cell surface retention (33), but CASR does not contain the -PPSPFR- epitope, present near the carboxyl terminus of group I mGluRs, which is required for interaction with the Homer EVH1 domain (34).
In summary, studies in ß-arrestin2 null mice indicate the importance of CASR desensitization in the regulation of PTH secretion. In the parathyroid gland, GRK4 appears to act primarily through the classical pathways to promote ß-arrestin uncoupling of CASR signaling, whereas GRK2 has in addition postreceptor actions that are likely related to its RGS-like interactions with Gq. The fact that GRK4 but not GRK2 supports translocation of ß-arrestin-GFP to CASR suggests important characteristics of CASR that may serve to limit the duration of desensitization and thereby maintain responsiveness of CASR to rapid changes in extracellular calcium.
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MATERIALS AND METHODS |
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Assessment of PTH Levels in ß-Arrestin2-Deficient Mice
ß-Arrestin2-deficient mice that had been back-crossed for at least six generations onto the C57/BL6 background were used for these studies (37). ß-Arrestin2-deficient mice were genotyped by using PCR on DNA samples prepared from tail tips, as previously described (38). Serum PTH levels were measured by mouse intact PTH ELISA kit (Immutopics, Carlsbad CA) as previously described (39). Calcium was measured by the colorimetric cresolphthalein-binding method (40), and ionized calcium was measured by a calcium-specific electrode (Bayer Rapidlab 865, Bayer AG, Leverkusen, Germany). To assess hypocalcemia-induced stimulation of PTH, wild-type and ß-arrestin2 null mice were administered a single ip injection of 300 µM/kg body weight of EGTA in saline or treated with saline only as controls (41). Serum was collected for each mouse 30 min after administration of the EGTA by eye bleeding. Animal studies were approved by the Laboratory Animal Resources, University of Kansas Medical Center.
Cell Culture
HEK-293 and COS-7 cells were grown in DMEM (QIAGEN, Inc., Valencia, CA) supplemented with 10% fetal calf serum, 1% penicillin/streptomycin at 37 C in a humidified atmosphere of 95% air/5% CO2, and incubated in DMEM containing 0.1% BSA, 1% penicillin/streptomycin, and 0.5 mM Ca+2. Human osteosarcoma cells (U2OS) were purchased from the ATCC and grown in DMEM supplemented with 10% (vol/vol) heat-inactivated fetal calf serum and gentamicin (100 µg/ml). The generation of U2OS cells stably expressing ß-arrestin2-GFP has been described previously (42).
Sources and Construction of Expression Plasmids
The rat CASR cDNA was obtained from Drs. A. M. Snowman and S. H. Snyder (43) and subcloned in the mammalian expression vector pcDNA 3 (Invitrogen, Carlsbad, CA) as previously described (35). We used the previously described SRE-luciferase plasmid DNA for reporter assays (44). SRE-luciferase activity provides a convenient readout for CASR activation and correlates with other measures of intracellular signaling downstream of CASR (31). Mutually priming oligonucleotides and PCR were used to insert the FLAG tag in the N terminus of CASR immediately 3' to the signal peptide (45). The PCR products were digested with HindIII and XbaI, purified, and ligated into a modified expression vector pSV.SPORT. pcDNA3.FLAG.ß-arrestin1 and ß-arrestin2, and pRK5.GRK2, and GRK3, and GRK4 and GRK5 were a obtained from Dr. R. J. Lefkowitz (46). Construction of the ß-arrestin2-GFP expression vector has been described previously (42).
Transfection
All plasmid DNAs were prepared using the EndoFree Plasmid Maxi Kit (Invitrogen). Transient transfections were preformed as follows: 2 x 105 HEK-293 or HEK-293 cells stably transfected with CASR were plated in the six-well plate and incubated overnight at 37 C. A DNA-liposome complex was prepared by mixing DNA of the SRE-luciferase reporter plasmid, pCMV-ß-gal, and other expression vectors as indicated with TransFast transfection reagent (Promega, Madison, WI). The total plasmid DNA was equalized in each well by adjusting the total amount of DNA to 2 µg/well with the empty vector.
Assessment of Agonist-Stimulated SRE Activity
Quiescence of transfected cells was achieved in subconfluent cultures by removing the media and washing twice with Hanks’ balanced salt solution to remove residual serum, followed by incubation for an additional 24 h in serum free DMEM containing 0.1% BSA. Luciferase activity was assessed after 8 h of stimulation. The luciferase activity in cell extracts was measured using the luciferase assay system (Promega) following the manufacturer’s protocol using a BG-luminometer (Gem Biomedical Inc., Sparks, NV).
Immunoprecipitations
After cotransfection with FLAG-tagged ß-arrestin1 and pcDNA3.0-rCASR, HEK-293 cells were rinsed with PBS and lysed in immunoprecipitation buffer. Cell lysates were precipitated with mouse anti-FLAG M2 monoclonal antibody and protein A-Sepharose beads (Sigma). For controls, the above procedure was duplicated without the addition of the antibody or using another mouse monoclonal antibody V5. The precipitates were separated by 6% SDS-PAGE and then immunoblotted with mouse anti-CASR antibody ADD (1/32,000) (NPS Pharmaceuticals, Inc., Salt Lake City, UT) and detected by ECL+Plus (Amersham Pharmacia Biotech, Piscataway, NJ).
Agonist-Dependent Internalization Assays
A BamHI fragment containing the entire rat CASR coding sequence was subcloned into the mammalian expression vector pcDNA3 (Invitrogen). The orientation and the nucleotide sequence of CASR were confirmed by sequencing both the 5' and 3' ends of the insert. To create the CASR tagged at the amino terminus with the FLAG epitope, we used the technique of mutually priming oligonucleotides to insert the FLAG epitope into the amino terminus of CASR after the signal peptide sequence. HEK-293 cells expressing the FLAG-tagged CASR were incubated with 5 mM Ca+2 for 5, 10, or 30 min at 25 C, and cell-surface expression of epitope-tagged CASR was assessed by flow cytometry using anti-FLAG antibody (1:500) (Sigma) as the first antibody and a goat antimouse IgG antibody conjugated to fluorescein isothiocyanate (1:500) (ICN Pharmaceuticals, Inc., Costa Mesa, CA) as the second antibody. HEK-293 cells were also transiently transfected with FLAG-ß2AR, and internalization was assessed by modifications of previously described methods (47).
Receptor Phosphorylation
FLAG-tagged CASR-expressing HEK-293 cells were labeled with 32P (0.1–0.2 mCi) (NEN Life Science Products, Boston, MA) and stimulated with agonist for 10 min at the indicated concentrations. CASR was immunoprecipitated using the M2 monoclonal antibody (Sigma) and proteins separated by SDS-PAGE. The phosphorylated proteins were detected by autoradiography.
Mammalian Two-Hybrid Analysis
We used a Mammalian Two-Hybrid assay system (Invitrogen) to evaluate CASR and ß-arrestins binding. We created the bait linear cDNA Psv40/VSL4/CASR (either CASR 877-1079 or CASR 636–805) and the prey linear cDNA Psv40.VP16/ß-arrestins (full-length ß-arrestin1 and 2) using a PCR approach with primers containing gene-specific sequences and TOPO Tools-specific overhangs (Invitrogen). COS-7 cells were transfected using TransFast reagent (Promega) with a respective prey/bait/reporter ratio of 0.5 µg/0.5 µg/1 µg (total DNA 2 µg/well). Reporter activity was assayed using ß-Gal Assay Kit according to the manufacturer’s instructions (Invitrogen).
Confocal Microscopy
The detailed methods for redistribution of fluorescently labeled arrestins from the cytoplasm to agonist-occupied receptors at the plasma membrane have been described previously (42). Briefly, U2OS cells stably expressing ß-arrestin2-GFP were transiently transfected with CASR alone or with CASR and GRK2, GRK3, GRK4, GRK5, or GRK6. Transfections were performed with FuGENE 6 (Roche) and typically resulted in 10–15% transfection efficiencies. Transfected cells were plated on 35-mm glass-bottom dishes (MatTek, Ashland, MA) and cultured overnight. Two hours before analysis, the medium was removed and replaced with serum- and phenol red-free medium supplemented with 10 mM HEPES. Confocal microscopy was performed on a Zeiss laser scanning microscope (LSM 5 Pascal; Carl Zeiss, Jena, Germany). Images were taken from the bottom of live cells before and after CaCl2 treatment and were acquired in real time using single-line excitation (488 nm).
RT-PCR Analysis
We isolated RNA from human parathyroid and kidney cortical tissues by grinding snap-frozen tissues in liquid nitrogen and then extracting total RNA using Trizol reagent (Molecular Research Center, Inc., Cincinnati, OH). RNA samples, pretreated with DNase, were further cleaned using an RNeasy spin column (QIAGEN), and the yield was quantified using a Ribogreen RNA quantitation kit (Molecular Probes, Eugene, OR). To detect ß-arrestins and GRK expression in human parathyroid gland and kidney, RT-PCR was performed using a two-step RNA PCR procedure by modification of previously described methods (48). Briefly, in separate reactions, 2.0 µg of DNase-treated total RNA was reverse-transcribed into cDNA with the respective reverse primers specified below and Moloney murine leukemia virus reverse transcriptase (Invitrogen). Reactions were carried out at 42 C for 60 min, followed by 94 C for 5 min and 5 C for 5 min. The products of first strand cDNA synthesis were directly amplified by PCR using AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA) using three separate sets of primers based on the human ß-arrestins and GRK cDNA sequence. PCR was performed with thermal cycling parameters of 94 C for 3 min, 94 C for 1 min, 60 C for 1 min, and 72 C for 2 min for 35 cycles, followed by a final extension at 72 C for 10 min. The respective primer sets used to amplify human ß-arrestins and GRKs were: hßarr1.15F (5'-gacgcgagtgttcaagaagg-3') and hßarr1.1253R (5'-ctgttgttgagctg-tggagagc-3'); hßarr2.110F (5'-agggtcttcaagaagtcga-3') and hßarr2.1040R (5'-ctcgagacaccaccagcttcacc-3'); hGRK1.F (5'-gctttgacggcagcagc-3') and hGRK1.R (5'-gggttctcctcattcacg-3'); hGRK2.F (5'-agcccctttttccgctccc-3') and hGRK2.R (5'-ccgcgctggaccagcggcac-3'); hGRK3.F (5'-cacagctttttcaaaggtg-3') and hGRK3.R (5'-ttcctgtgacag-agggatgg-3'); hGRK4.F (5'-cctttaccagaaatacctcc-3') and hGRK4.R (5'-cttacagtaaacggcatgagg-3'); hGRK5.F (5'-gaaggaaattatgacc-3') and hGRK5.R (5'-ctccgtctccaggacc-3'); hGRK6.F (5'-tcacagcctgtgcgagcg-3') and hGRK1.R (5'-gttcggcagggtccttgc-3'); hGRK7.F (5'-catggctttcttgcaagagc-3') and hGRK7.R (5'-tgtggttgtgatgtccc-3'). Human glyceraldehyde-3-phosphate dehydrogenase was amplified as a control. Amplification products were resolved by electrophoresis on a 1.0% agarose gel and visualized by ethidium bromide staining.
Statistics
We evaluated differences between groups by one-way ANOVA. Values sharing the same superscript are not significantly different at P < 0.05. All computations were performed using the Statgraphic statistical graphics system (STSC, Inc., Princeton, NJ).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online January 6, 2005
Abbreviations: CASR, Calcium-sensing receptor; GABA, -aminobutyric acid; GPCR, G protein-coupled receptors; GRK, G protein receptor kinase; HEK, human embryonic kidney; RGS, regulators of G protein signaling; SRE, serum response element.
Received for publication November 8, 2004. Accepted for publication December 27, 2004.
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