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Endocrinology, doi:10.1210/en.2006-1035
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Endocrinology Vol. 148, No. 5 2398-2404
Copyright © 2007 by The Endocrine Society

Functional Desensitization of the Extracellular Calcium-Sensing Receptor Is Regulated via Distinct Mechanisms: Role of G Protein-Coupled Receptor Kinases, Protein Kinase C and ß-Arrestins

Stephan Lorenz1, Romy Frenzel1, Ralf Paschke, Gerda E. Breitwieser and Susanne U. Miedlich

III. Medical Department (S.L., R.F., R.P.), Leipzig University, D-04109 Leipzig, Germany; Weis Center for Research (G.E.B.), Geisinger Clinic, Danville, Pennsylvania 17822; and Endocrine Unit (S.U.M.), Massachusetts General Hospital, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Susanne U. Miedlich, Endocrine Unit, Massachusetts General Hospital, Bulfinch 327, 55 Fruit Street, Boston, Massachusetts 02114. E-mail: smiedlich{at}partners.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The extracellular calcium-sensing receptor (CaR) senses small fluctuations of the extracellular calcium (Ca2+e) concentration and translates them into potent changes in parathyroid hormone secretion. Dissecting the regulatory mechanisms of CaR-mediated signal transduction may provide insights into the physiology of the receptor and identify new molecules as potential drug targets for the treatment of osteoporosis and/or hyperparathyroidism. CaR can be phosphorylated by protein kinase C (PKC) and G protein-coupled receptor kinases (GRKs), and has been shown to bind to ß-arrestins, potentially contributing to desensitization of CaR, although the mechanisms by which CaR-mediated signal transduction is terminated are not known. We used a PKC phosphorylation site-deficient CaR, GRK and ß-arrestin overexpression or down-regulation to delineate CaR-mediated desensitization. Fluorescence-activated cell sorting was used to determine whether receptor internalization contributed to desensitization. Overexpression of GRK 2 or 3 reduced Ca2+e-dependent inositol phosphate accumulation by more than 70%, whereas a GRK 2 mutant deficient in G{alpha}q binding (D110A) was without major effect. Overexpression of GRK 4–6 did not reduce Ca2+e-dependent inositol phosphate accumulation. Overexpression of ß-arrestin 1 or 2 revealed a modest inhibitory effect on Ca2+e-dependent inositol phosphate production (20–30%), which was not observed for the PKC phosphorylation site-deficient CaR. Agonist-dependent receptor internalization (10–15%) did not account for the described effects. Thus, we conclude that PKC phosphorylation of CaR contributes to ß-arrestin-dependent desensitization of CaR coupling to G proteins. In contrast, GRK 2 predominantly interferes with G protein-mediated inositol-1,4,5-trisphosphate formation by binding to G{alpha}q.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PRECISE CONTROL OF the extracellular calcium concentration [Ca2+e] between 2.2–2.4 mmol/liter is essential for many physiological functions, including contraction of skeletal and smooth muscle cells, neuronal transmission, and blood coagulation. Small fluctuations of [Ca2+e] induce potent changes in parathyroid hormone secretion and subsequent calcium absorption or elimination by intestines, kidneys, and bones (1), presumably via activation of the calcium-sensing receptor (CaR). The precise translation of alterations of [Ca2+e] into changes in parathyroid hormone secretion makes CaR a promising target for therapeutic drug design. Allosteric activators of CaR have been approved for the treatment of refractory secondary hyperparathyroidism associated with progressive renal failure (2). Allosteric inhibitors are being tested in animal studies for the treatment of osteoporosis (3). In addition to the regulation of parathyroid hormone secretion, activation of the widely expressed CaR regulates various other cellular functions such as proliferation, differentiation, and apoptosis (4, 5). Dissecting the regulatory mechanisms of CaR-mediated signal transduction may not only provide insights into the physiology of the receptor but will identify critical interacting proteins as potential drug targets for treating diseases of mineral and bone metabolism as well as cancers of colon or breast.

Family C G protein-coupled receptors (GPCRs), including CaR, GABAB, and metabotropic glutamate receptors are characterized by an unusually large extracellular domain that resembles the venus-flytrap domain of bacterial periplasmic binding proteins and serves as the site for agonist binding (6). CaR couples to the heterotrimeric Gq, Gi, and G12/13 proteins and regulates numerous second messengers such as inositol-1,4,5-trisphosphate, Ca2+i, cAMP, and phosphatidic acid (7). Activation of MAPK is also mediated by CaR and is dependent upon its interaction with filamin A, a large actin-binding protein that integrates scaffold and signaling functions (8, 9, 10).

Agonist-dependent activation of GPCRs is generally followed by initiation of regulatory mechanisms leading to rapid signal attenuation, termed functional desensitization. Receptor phosphorylation by GPCR kinases (GRKs) and/or second messenger-dependent protein kinases [protein kinase C (PKC) or protein kinase A] is regarded as an early step in functional desensitization. Subsequently, ß-arrestins, which exhibit a high affinity for the activated and/or phosphorylated GPCR, mediate uncoupling from G proteins. Association of the receptor-ß-arrestin complex with clathrin initiates receptor internalization that can also contribute to functional desensitization (11). Independent of receptor phosphorylation, GRKs can mediate signal attenuation by direct binding to the activated form of G{alpha} proteins. The latter process is mediated by a regulator of G protein signaling (RGS)-homology domain and has been described for GRK 2, 3, and 4 (12, 13).

CaR can be phosphorylated by PKC, GRKs 2 and 4, and has been shown to bind to ß-arrestins, likely contributing to functional desensitization of CaR (14, 15). However, the precise mechanisms by which second messenger-dependent kinases, GRKs, and/or ß-arrestins terminate CaR-mediated signal transduction are not known. In this report, we use a PKC phosphorylation site-deficient CaR, GRK, and ß-arrestin overexpression or down-regulation to demonstrate that GRK 2 predominantly interferes with inositol-1,4,5-trisphosphate accumulation by binding to G{alpha}q, thereby reducing PLCß activity. In contrast, elimination of PKC phosphorylation sites but not GRK 2 down-regulation abolishes ß-arrestin-dependent desensitization of CaR signaling to Gq proteins. CaR internalization does not contribute significantly to functional desensitization of Gq signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mutagenesis, cell culture, and transfection
All five of the predicted PKC phosphorylation sites of CaR were mutated to alanine in the background (fusion proteins previously described in Refs. 16 and 17) by site-directed mutagenesis and confirmed by restriction endonuclease digestion and direct sequencing. A Flag-CaR construct was used for cell sorting analysis (10). ß-Arrestin 1 and 2 and GRK 3, 5, and 6 constructs were generous gifts from Dr. J. L. Benovic (Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA). GRK2, GRK2-D110A, GRK2-K220R, and GRK2-D110A/K220R were kindly provided by Dr. S. S. Ferguson (Robarts Research Institute, London, Ontario, Canada). Generation of the GRK 4 construct has been described elsewhere (18). GripTite 293 MSR cells (which stably express the macrophage scavenger receptor to increase adherence; Invitrogen Group, Paisley, UK) were grown in high glucose DMEM (PAA Laboratories, Pasching, Austria) supplemented with 10% fetal bovine serum (PAA Laboratories), nonessential amino acids (0.1 mM; PAA Laboratories), and geneticin (600 µg/ml; Life Technologies, Inc., Invitrogen) at 37 C, 5% CO2. For transient transfections, cDNA of CaR plus the respective ß-arrestin or GRK construct (or empty vector, pcDNA3.1) at a 4:1 ratio (overall 1 µg cDNA per well, 12-well plate), GeneJammer (Stratagene Corp., La Jolla, CA), and medium were premixed according to manufacturer’s protocols, added to the cells (50% confluent), and supplemented with regular medium. Experiments were carried out 48–72 h after transfection. Transfections with STEALTHsiRNA against GRK 2 (19, 20), ß-arrestins, or control small interfering RNA (siRNA; Invitrogen Group) were carried out 24 h after transfection with receptor (and control plasmid or respective ß-arrestin). siRNA (300 ng/well, six-well plate, after initial titration by Western blotting), HiPerFect (QIAGEN GmbH, Hilden, Germany), and 100 µl DMEM were premixed and subsequently added to the cells. Experiments were carried out 48 h after the second transfection.

Immunoprecipitation
Transfected GripTite 293 cells were briefly washed with PBS containing 1 mM EDTA, suspended in lysis buffer (5 mM EDTA, 100 mM iodoacetamide, 0.5% Triton X-100 in PBS) with protease inhibitor mix (Cømplete; Roche Applied Sciences, Mannheim, Germany) and gently agitated for 30 min. The samples were preincubated with 25 µl protein A agarose (Invitrogen Group) to minimize nonspecific binding and centrifuged at 14,000 rpm for 2 min, and the supernatant was used for immunoprecipitation. Two microliters of a polyclonal G{alpha}q protein antibody solution (Cell Signaling Technology Inc., Danvers, MA) were added to the supernatant and incubated for 2 h at 4 C, incubation was continued for 18 h after addition of 25 µl of protein A agarose. The resin was washed three times with lysis buffer and incubated with 30 µl of Western blot loading buffer (12 M urea, 4% SDS, 0.01% bromphenol blue, 100 mM ß-mercaptoethanol in 200 mM Tris) for 30 min at room temperature. Samples were separated on 4–15% Tris-HCl SDS polyacrylamide gels as described below.

Western blotting
Transfected GripTite 293 cells were briefly washed with PBS containing 1 mM EDTA, suspended in lysis buffer (5 mM EDTA, 100 mM iodoacetamide, 0.5% Triton X-100 in PBS) with protease inhibitor mix (Cømplete; Roche Applied Sciences) and gently agitated for 30 min. Supernatants were assayed for protein (Pierce Biotechnology, Rockford, IL) according to the manufacturer’s protocol. Equal amounts of protein were separated on 10% Tris-HCl SDS gels and blots probed with antibodies against ß-arrestin 1 (Cell Signaling Technology Inc.), GRK 2–6 (Santa Cruz Biotechnology, Inc., Heidelberg, Germany), or CaR (AffinityBioReagents Inc., Golden, CO) at 4 C overnight. After incubation with polyclonal anti-HRP antibody (1:3000), blots were developed with Super Signal West Pico (Pierce Biotechnology).

Inositol phosphate assay
Inositol phosphates were measured as previously described (21). Briefly, 48–72 h after transfection, GripTite 293 cells were incubated with 2 µCi/ml myo-[3H]inositol (18.6 Ci/mmol; Amersham plc, Buckinghamshire, UK) in regular medium for 8 h. Subsequently, cells were stimulated with variable Ca2+e solutions for 1 h (plus 1 mM LiCl2, 140 mM NaCl, 5 mM KCl, 0.55 mM MgCl2, 10 mM HEPES, pH 7.4). Variations of Ca2+e were produced by compensatory changes for NaCl to maintain constant solution osmolality. Stimulation was terminated by aspiration and addition of 3% perchloric acid. Inositol phosphate levels were determined by anion exchange chromatography and liquid scintillation counting, and are expressed as the percentage of total radioactivity incorporated in inositol phosphates and phosphatidyl inositols.

Fluorescence-activated cell sorting (FACS) analysis
To determine cell surface expression of CaR (Flag-CaR), transfected cells were incubated with a polyclonal anti-Flag antibody (Sigma-Aldrich) 1:1000 in PBS containing 1% BSA and 1% sodium azide at 4 C. After two washes with PBS (1% BSA, 1% sodium azide), cells were incubated with Alexa Fluor 546-conjugated goat antirabbit IgG antibody (Molecular Probes, Invitrogen Group) 1:800 in PBS (1% BSA, 1% sodium azide) for 1 h in the dark (4 C). After two final washes, cells were fixed with 1% paraformaldehyde. The fluorescence of 10,000 cells per tube was assayed with a FACSScan cytofluorometer (Becton Dickinson, San Jose, CA).

Data analysis
Inositol phosphate (inositol phosphate assay) and fluorescence (FACS analysis) values calculated as described in the previous sections were normalized to the control condition within each experimental set and subsequently used for statistical analysis. All statistical/graphical analyses were done with SPSS 10.0 (SPSS Inc., Chicago, IL), SigmaPlot 2000 for Windows version 6.0 (SPSS Inc.), and GraphPad PRISM 3.02 (GraphPad Inc., San Diego, CA). Because the majority of the data were normally distributed according to Kolmogorov Smirnov tests, one-way ANOVA and post hoc t tests were used to compare experimental conditions using the different ß-arrestin/GRK constructs. Curves were fitted using classic equations for sigmoidal concentration-responses with variable slopes (GraphPad PRISM 3.02). A P value of less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Homologous desensitization of GPCRs is generally mediated by GRKs through phosphorylation-dependent and independent mechanisms (12). GRKs 2 and 4 can phosphorylate CaR (15) and, along with ß-arrestins, can attenuate CaR-mediated signaling (15). We used inositol phosphate assays to characterize the mechanisms of CaR functional desensitization. First, we determined the effects of nonvisual GRKs (GRK 2–6) on CaR-mediated inositol phosphate accumulation. GripTite 293 cells were transiently transfected with wild-type CaR and a control plasmid (pcDNA3.1), or GRK 2–6 (ratio of transfected CaR to GRK cDNAs was 4:1). Forty-eight to 72 h after transfection, Western blots or inositol phosphate assays were carried out as described in Materials and Methods. GripTite 293 cells endogenously express GRKs 2, 5, and 6 (GRK 6 > GRK 2 > GRK 5). Transient transfection with GRK 2–6 resulted in comparable increases of protein levels as determined by Western blotting (Fig. 1BGo). Overexpression of cytosolic GRK 2 or 3 reduced CaR-mediated inositol phosphate accumulation in response to 10 mM Ca2+e by approximately 72.16 ± 9.61% or 84.59 ± 2.6%, respectively (P < 0.001; Fig. 1AGo). Overexpression of membrane-localized GRK 4, 5, and 6 had no effect on CaR-mediated inositol phosphate accumulation.


Figure 1
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  		FIG. 1. A, Effects of GRK overexpression on CaR-mediated inositol phosphate (IP) formation. Cells were transiently transfected with CaR and pcDNA or GRK 2–6 (ratio of CaR to GRK was 4:1) and subjected to inositol phosphate assays 48–72 h later. Normalized inositol phosphate responses (%) were determined as inositol phosphate formation in 10 mM Ca2+e, minus inositol phosphate formation in 0.5 mM Ca2+e, normalized to the control condition (CaR plus pcDNA, black column). ***, P < 0.001. Mean inositol phosphate formation for the control condition at 0.5 mM Ca2+ was 4.5 ± 2.2%, and at 10 mM Ca2+ was 41.9 ± 6.2%. B, GRK protein (65–80 kDa) expression in untransfected (lane 1) or transfected (as above, lane 2) GripTite 293 cells. Representative data of two independent transfections are shown.

 
GRK 2 and 3 are members of the ß-adrenergic receptor kinase subfamily. These cytosolic kinases translocate to the membrane by binding to Gß{gamma} subunits released upon G protein activation and subsequently phosphorylate the activated GPCR. In addition to the central catalytic kinase domain, the amino-terminal domain of GRKs 2 and 3 contains a region of homology to RGS proteins that binds to activated G{alpha}q subunits and inhibits phospholipase Cß stimulation (22, 23). We used previously described mutants of GRK 2 (22, 24) to determine whether kinase activity and/or G{alpha}q binding contributes to inhibition of CaR signaling by ß-adrenergic receptor kinases. GripTite 293 cells were transiently transfected with CaR and pcDNA3.1, wild-type GRK 2, or a GRK 2 mutant defective in kinase activity (K220R) and/or in G{alpha}q binding (D110A). Inositol phosphate accumulation was measured after CaR stimulation with either 0.5 or 10 mM Ca2+e. Surprisingly, the catalytic site mutant of GRK 2 (K220R) did not differ significantly from wild-type GRK 2 in its ability to inhibit CaR signaling. In contrast, the G{alpha}q binding site mutant of GRK 2 (D110A) inhibited the inositol phosphate signal significantly less than wild-type GRK 2 (P < 0.001; Fig. 2Go); similar results were observed with the double mutant (D110A/K220R). We conclude that GRK 2 predominantly inhibits CaR signaling by direct interaction with G{alpha}q, whereas kinase activity is mostly redundant for agonist-dependent functional desensitization of CaR. Although, the mutant GRK 2 D110A still inhibited inositol phosphate formation by 22.3 ± 3.31% (P < 0.001) when compared with the control condition. This residual effect of the GRK 2 mutant may be attributed to sterical interference of the GRK with G proteins at the receptor level. Thus, we coimmunoprecipitated endogenous G{alpha}q proteins and CaR from cells transfected with the receptor and pcDNA or GRK 2 as above. Overexpression of GRK 2 negatively regulated the interaction between CaR and G{alpha}q (see Fig. 2BGo) suggesting that GRKs may indeed interfere with G proteins at the receptor level.


Figure 2
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  		FIG. 2. A, Effects of wild-type or mutant GRK 2 overexpression on CaR-mediated inositol phosphate (IP) formation. Inositol phosphate responses (%) were determined as in Fig. 1Go and normalized to the control condition (CaR plus pcDNA, black column). Overexpression of GRK 2 wild-type (white column) was compared with overexpression of distinct GRK 2 mutants (gray columns): D110A is defective in G{alpha}q binding, K220R is defective in kinase activity, double = D110A, K220R. ***, P < 0.001. B, Interaction of G{alpha}q proteins and CaR is negatively affected by GRK 2 overexpression. GripTite 293 cells transiently transfected with CaR plus pcDNA (lanes 3 and 4) or GRK 2 (lanes 5 and 6) were subjected to coimmunoprecipitation of G{alpha}q proteins after a 1-h incubation in 0.5 (lanes 1, 3, and 5) or 10 mM Ca2+ (lanes 2, 4, and 6) at 37 C. Immunoprecipitates (lower panel) and lysates (upper panel) were subsequently blotted for CaR. Untransfected cells served as negative controls (lanes 1 and 2). Representative data of two independent transfections are shown.

 
We next investigated the effects of ß-arrestin overexpression on CaR-mediated inositol phosphate production. CaR plus ß-arrestin 1 or 2 (ratio of transfected CaR to ß-arrestin cDNAs was 4:1) were transfected into GripTite 293 cells. ß-Arrestin 1 or 2 inhibited inositol phosphate accumulation in response to CaR stimulation with 10 mM Ca2+e (CaR plus ß-arrestin 1: 21.03 ± 5.0%, P < 0.05 compared with control; CaR plus ß-arrestin 2: 27.32 ± 6.29%, P < 0.01 compared with control; Fig. 3AGo). On the contrary, down-regulation of both ß-arrestins by RNA interference (approximately 60% reduction for ß-arrestin 1 siRNA, 80% reduction for ß-arrestin 2 siRNA, data not shown), which are both expressed in GripTite 293 cells increased inositol phosphate formation in response to 10 mM Ca2+e (see supplemental data).


Figure 3
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  		FIG. 3. A, Effects of ß-arrestin overexpression on CaR-mediated inositol phosphate (IP) formation. Normalized inositol phosphate responses (%) were determined as in Fig. 1Go and normalized to the control condition (CaR plus pcDNA). Data sets represent CaR-mediated inositol phosphate responses using either control plasmid (pcDNA, black columns) or ß-arrestin 1 (white columns) or 2 (gray columns). B, GRK 2 down-regulation by specific siRNA. Cells were transfected with siRNA against GRK 2 or control siRNA, and cells lysates were subjected to Western blotting for GRK 2 expression. Representative data of three independent transfections are shown. C, GRK 2 down-regulation by RNA interference does not significantly affect ß-arrestin-dependent reduction of inositol phosphate accumulation. CaR-mediated responses were obtained as in A, 48 h after additional transfection with respective siRNAs. Control conditions within each data set were compared with conditions overexpressing ß-arrestin 1 or 2. *, P < 0.05; **, P < 0.01.

 
GRK-dependent receptor phosphorylation facilitates ß-arrestin binding to many GPCRs and may also mediate ß-arrestin-dependent CaR desensitization. Therefore, we evaluated whether ß-arrestin-dependent attenuation of CaR signaling would be modified by down-regulation of GRK 2 expression by RNA interference. GRK 2 but not GRK 3 is well expressed in GripTite 293 cells as determined by Western blotting (see Fig. 1BGo), and siRNA against GRK 2 but not control siRNA caused a concentration-dependent reduction in GRK 2 to undetectable levels 48 h after transfection (Fig. 3BGo). Surprisingly, the inhibitory effects of ß-arrestins on CaR signaling were not significantly modified by down-regulation of GRK 2 (P > 0.2; Fig. 3CGo). Taken together, these results strongly suggest that GRK 2 inhibits CaR-mediated inositol phosphate accumulation independent of receptor phosphorylation and ß-arrestin binding.

Consensus sequence mapping of the human CaR identified five putative PKC phosphorylation sites (14). It has previously been shown that CaR is phosphorylated by PKC (14). In addition, elimination of all putative PKC phosphorylation sites significantly reduced receptor phosphorylation by PKC as well as functional desensitization after PKC activation (14). We generated a CaR mutant with alanine residues at all putative PKC phosphorylation sites (T646A, S794A, T888A, S895A, S915A = CaR/no PKC site) and determined whether ß-arrestin-dependent desensitization was affected. ß-Arrestin-dependent reduction of stimulated inositol phosphate accumulation was not observed for the PKC phosphorylation-deficient CaR (CaR/noPKC site plus control, 103.52 ± 10.67%; CaR/noPKC site plus ß-arrestin 1, 116.53 ± 11.51%; CaR/noPKC site plus ß-arrestin 2, 108.84 ± 6.24%; P > 0.5; Fig. 4Go). To confirm this result, we transfected wild-type CaR and ß-arrestins and determined stimulated inositol phosphate accumulation in the absence and presence of the specific PKC inhibitor GF109203X (1 µM). ß-Arrestin-dependent functional desensitization after stimulation with 10 mM Ca2+e was not observed in the presence of the PKC-inhibitor (CaR/PKC inhibitor plus control, 121.1 ± 16.25%; CaR/PKC inhibitor plus ß-arrestin 1, 111.37 ± 12.72%; CaR/PKC inhibitor plus ß-arrestin 2, 99.05 ± 14.85%; P > 0.5; Fig. 4Go).


Figure 4
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  		FIG. 4. Effects of ß-arrestin overexpression on inositol phosphate (IP) responses are abolished by eliminating PKC phosphorylation sites of CaR. Normalized inositol phosphate responses (%) were determined as in Fig. 1Go and normalized to the control condition (CaR plus pcDNA). Data sets represent inositol phosphate responses using cells transfected with either CaR wild-type in the absence or presence of a PKC inhibitor (1 µM GF109203X) or the non-PKC-phosphorylatable CaR (CaR/no PKC site) plus control plasmid (pcDNA, black columns) or ß-arrestin 1 (white columns) or 2 (gray columns). Control conditions within each data set were compared with conditions overexpressing ß-arrestin 1 or 2. *, P < 0.05; **, P < 0.01.

 
The Ca2+e concentration response relationship obtained after transient transfection of GripTite 293 cells with either wild-type or the non-PKC-phosphorylatable CaR confirmed a significantly lower EC50 of the mutant compared with the wild-type receptor (1.2 vs. 3.1 mM Ca2+e; Fig. 5AGo). Similar results were obtained in parental HEK 293 cells transfected with CaR (Miedlich, S. U., unpublished observations, and Ref. 14). Concentration responses obtained for wild-type and non-PKC-phosphorylatable CaR plus ß-arrestin 1 or 2 confirmed a slight right shift and a decrease in the maximal response for the wild-type but not for the non-PKC-phosphorylatable CaR (Fig. 5Go, B and C), consistent with the results obtained in Fig. 4Go. Vice versa, down-regulation of ß-arrestin 1 and 2 expression by specific siRNAs led to an increase of the maximal response for the wild-type receptor (124.5 ± 6.53%, see supplemental data).


Figure 5
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  		FIG. 5. Ca2+e-dependent concentration response curves of CaR-mediated inositol phosphate (IP) formation as a function of PKC-dependent receptor phosphorylation and ß-arrestin overexpression. Curves were fitted with GraphPad PRISM 3.02, using the following equation: Y = Rbas + (Rmax – Rbas)/(1 + 10^((logEC50 – X)*n)). A, CaR wild-type (bullet): EC50 = 3.07 mM, n = 2.13, Rmax = 60.2%; CaR/no PKC site ({circ}): EC50 = 1.24 mM, n = 1.91, Rmax = 63.7%. B, Wild-type CaR plus ß-arrestin 1 ({blacktriangleup}): EC50 = 3.45 mM, n = 2.88, Rmax = 47.7%; wild-type CaR plus ß-arrestin 2 ({blacktriangledown}): EC50 = 3.87 mM, n = 2.66, Rmax = 48.8%. C, Non-PKC-phosphorylatable CaR plus ß-arrestin 1 ({triangleup}): EC50 = 1.66, n = 2.41, Rmax = 61.6%; non-PKC-phosphorylatable CaR plus ß-arrestin 2 ({triangledown}): EC50 = 1.52, n = 2.22, Rmax = 65.99%.

 
Receptor internalization may also contribute to functional desensitization of CaR. Cell surface CaR was quantified by FACS analysis of cells expressing Flag-tagged CaR. DMEM contains 1.8 mM Ca2+e, a concentration just below the EC50 of CaR (3.1 mM), which may induce stimulation-dependent internalization and/or down-regulation of CaR. To avoid receptor stimulation and potential internalization before the experiment, cells were incubated for 12 h before the experiments in HAM’s F12 medium containing 0.33 mM Ca2+ (PAA Laboratories). Incubation of CaR-expressing GripTite 293 cells in 10 mM Ca2+e for 60 min (at 37 C) did not reveal significant differences in receptor expression depending on exposure to Ca2+e (data not shown). In contrast, when cell surface receptors were prelabeled with polyclonal anti-Flag antibody (1:1000, in PBS plus 1% BSA, 1% sodium azide for 30 min, at 4 C), followed by incubation of the cells with 10 mM Ca2+e (at 37 C), we observed a small but significant loss of cell surface receptors (10–15%), which was maximal after 20 min and did not change thereafter (Fig. 6Go). Incubation with 0.5 mM Ca2+e (at 37 C) did not result in significant changes of receptor expression (data not shown). Overexpression of GRK 2 or 3, ß-arrestin 1 or 2 did not significantly alter this pattern (data not shown). Therefore, CaR shows only minor agonist-dependent internalization that is not significantly affected by GRK or ß-arrestin overexpression. These results suggest that internalization of CaR is not dependent on GRK phosphorylation or receptor interaction with ß-arrestins.


Figure 6
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  		FIG. 6. Ca2+e-dependent cell surface expression of CaR measured by FACS analysis. Transfected cells were preincubated in 0.33 mM Ca2+e for 12 h at 37 C, prelabeled with a polyclonal anti-Flag antibody at 4 C, and stimulated with 10 mM Ca2+e for 0–60 min (at 37 C). Fluorescence was normalized to the basal CaR cell surface expression at 0 min. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with basal condition (time = 0).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Functional desensitization of GPCRs represents an important regulatory mechanism mediated by receptor phosphorylation by GPCR- and/or second messenger-dependent kinases, ß-arrestin binding, and uncoupling from heterotrimeric G proteins as well as receptor internalization. The ß-adrenergic GPCR kinases (GRKs 2 and 3) can also attenuate intracellular signaling by direct interactions with G{alpha}q and/or Gß{gamma} proteins. Interaction with G{alpha}q occurs at the amino-terminal domain of GRKs that contains a region of homology to RGS proteins. GRKs 2 and 3 have been shown to interact with G{alpha}q via this domain promoting phosphorylation-independent attenuation of GPCR signaling (12). In this study, we demonstrate that overexpression of GRK 2 or 3 inhibits CaR-mediated inositol phosphate accumulation by more than 70% and, therefore, may contribute to CaR functional desensitization. GRK 2 mutants reveal that CaR signaling can be notably restored by preventing kinase binding to G{alpha}q. Surprisingly, the kinase activity of GRK 2 is not required for attenuation of agonist-dependent inositol phosphate accumulation. Similar results have been obtained for the metabotropic glutamate receptor 1a (mGluR 1a), which also couples to G{alpha}q (22, 23, 24). Thus, GRK 2 may rapidly terminate a subset of CaR signaling pathways dependent upon G{alpha}q without requiring receptor phosphorylation, ß-arrestin recruitment, or subsequent receptor internalization, although overexpression of ß-arrestins reduces maximal CaR-mediated inositol phosphate accumulation by 20–30%.

Pi et al. (15) investigated the effects of GRK overexpression on CaR signaling using a serum response element luciferase assay as a readout. Interestingly, they report a significant reduction of CaR signaling by GRK 2, 4, and ß-arrestins, which they attribute to both phosphorylation-dependent and -independent mechanisms. Serum response factor, which binds to and activates serum response element, can be activated by numerous second messengers, including MAPK and the Rho family of GTPases. Therefore, its regulation by CaR may involve multiple G protein-dependent and -independent pathways. Taken together, the results imply that GRKs 2 and 4 differentially modulate distinct CaR signaling cascades. Integration of the resulting signals may allow for individual cellular responses such as changes in parathyroid hormone secretion or cell growth and differentiation.

Surprisingly, our results suggest that the second messenger-dependent kinase PKC, and not GRK 2, mediates ß-arrestin binding and, thereby, ß-arrestin-dependent functional desensitization. In contrast, GRK-dependent functional desensitization is predominantly mediated by the inhibitory actions of GRK 2 on G{alpha}q proteins. In general, GRKs and ß-arrestins have been implicated in agonist-dependent, homologous desensitization of GPCRs, whereas second messenger-dependent kinases have been associated with agonist-independent, heterologous desensitization. To date, only a few receptors have been shown to undergo agonist-dependent functional desensitization and/or receptor internalization regulated by second messenger-dependent kinases (Frizzled4 receptor, cysteinyl leukotriene type 1 receptor, secretin receptor, thromboxane receptor, mGluR1a) (25, 26, 27, 28, 29, 30). Dopamine receptor internalization was induced by activation of PKC and was also dependent on ß-arrestins (31). PKC- and ß-arrestin-dependent internalization has also been shown for the Frizzled receptor with ß-arrestin recruitment requiring the adaptor protein, disheveled (25). In cells transfected with mGluR1a, a member of the same GPCR family as CaR, selective inhibition of PKC enhanced maximal glutamate-stimulated inositol phosphate accumulation by approximately 50% (29, 30), indicating that PKC induced functional desensitization of mGluR1a.

Overexpression of ß-arrestins slightly shifts the Ca2+e concentration response curve for wild-type CaR to the right and decreases the maximal response, probably reflecting decreased interactions of the receptor with heterotrimeric G proteins, likely because G proteins and ß-arrestins sterically compete for interaction with GPCRs (32). Consistent with reduced interactions between ß-arrestins and the PKC phosphorylation-deficient CaR, ß-arrestin-mediated changes in the Ca2+e concentration response curve are no longer observed.

In summary, our results indicate that functional desensitization of CaR is predominantly mediated by direct binding of GRK 2 to G{alpha}q proteins. Receptor activation and subsequent phosphorylation by PKC regulates attenuation of CaR signaling by ß-arrestins. Consequently, GRK 2-dependent desensitization of CaR signaling may specifically regulate G{alpha}q-dependent pathways, whereas PKC-dependent receptor phosphorylation and binding of ß-arrestins likely affects all downstream signaling cascades. It will be interesting to determine how these mechanisms translate into changes of parathyroid hormone secretion and/or gene transcription.


   Acknowledgments
 
We thank members of the Breitwieser lab for generation of the CaR constructs as well as Prof. M. Stumvoll for providing a productive environment at the Leipzig University.


   Footnotes
 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft to S.U.M. (680/3-2) and National Institutes of Health Grant GM58578 to G.E.B.

Disclosure Statement: The authors have nothing to declare.

First Published Online January 25, 2007

1 S.L. and R.F. contributed equally to this work. Back

Abbreviations: Ca2+e, Extracellular calcium; [Ca2+e], extracellular calcium concentration; CaR, calcium-sensing receptor; FACS, fluorescence-activated cell sorting; GPCR, G protein-coupled receptor; GRK, GPCR kinase; PKC, protein kinase C; RGS, regulator of G protein signaling; siRNA, small interfering RNA.

Received August 1, 2006.

Accepted for publication January 16, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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