Allosteric Modulation of the Calcium-sensing Receptor Rectifies Signaling Abnormalities Associated with G-protein α-11 Mutations Causing Hypercalcemic and Hypocalcemic Disorders*

Germline loss- and gain-of-function mutations of G-protein α-11 (Gα11), which couples the calcium-sensing receptor (CaSR) to intracellular calcium (Ca2+i) signaling, lead to familial hypocalciuric hypercalcemia type 2 (FHH2) and autosomal dominant hypocalcemia type 2 (ADH2), respectively, whereas somatic Gα11 mutations mediate uveal melanoma development by constitutively up-regulating MAPK signaling. Cinacalcet and NPS-2143 are allosteric CaSR activators and inactivators, respectively, that ameliorate signaling disturbances associated with CaSR mutations, but their potential to modulate abnormalities of the downstream Gα11 protein is unknown. This study investigated whether cinacalcet and NPS-2143 may rectify Ca2+i alterations associated with FHH2- and ADH2-causing Gα11 mutations, and evaluated the influence of germline gain-of-function Gα11 mutations on MAPK signaling by measuring ERK phosphorylation, and assessed the effect of NPS-2143 on a uveal melanoma Gα11 mutant. WT and mutant Gα11 proteins causing FHH2, ADH2 or uveal melanoma were transfected in CaSR-expressing HEK293 cells, and Ca2+i and ERK phosphorylation responses measured by flow-cytometry and Alphascreen immunoassay following exposure to extracellular Ca2+ (Ca2+o) and allosteric modulators. Cinacalcet and NPS-2143 rectified the Ca2+i responses of FHH2- and ADH2-associated Gα11 loss- and gain-of-function mutations, respectively. ADH2-causing Gα11 mutations were demonstrated not to be constitutively activating and induced ERK phosphorylation following Ca2+o stimulation only. The increased ERK phosphorylation associated with ADH2 and uveal melanoma mutants was rectified by NPS-2143. These findings demonstrate that CaSR-targeted compounds can rectify signaling disturbances caused by germline and somatic Gα11 mutations, which respectively lead to calcium disorders and tumorigenesis; and that ADH2-causing Gα11 mutations induce non-constitutive alterations in MAPK signaling.


G␣ 11 mutations were demonstrated not to be constitutively activating and induced ERK phosphorylation following Ca 2؉
o stimulation only. The increased ERK phosphorylation associated with ADH2 and uveal melanoma mutants was rectified by NPS-2143. These findings demonstrate that CaSR-targeted compounds can rectify signaling disturbances caused by germline and somatic G␣ 11 mutations, which respectively lead to calcium disorders and tumorigenesis; and that ADH2-causing G␣ 11 mutations induce non-constitutive alterations in MAPK signaling.
Guanine nucleotide-binding protein (G-protein) 4 ␣-11 (G␣ 11 ) is a major intracellular signaling partner of the cell-surface G-protein-coupled calcium (Ca 2ϩ )-sensing receptor (CaSR), which plays a pivotal role in the parathyroid and renal regulation of extracellular Ca 2ϩ (Ca 2ϩ o ) concentrations (1,2). G␣ 11 belongs to the G q/11 class of G-proteins that enhance phospholipase C activity (3), thereby leading to an accumulation of inositol 1,4,5-trisphosphate and rapid increase in intracellular Ca 2ϩ (Ca 2ϩ i ) concentrations (2,4). These signal transduction events allow the CaSR to respond to small fluctuations in the prevailing Ca 2ϩ o concentration ([Ca 2ϩ ] o ) by inducing alterations in parathyroid hormone (PTH) secretion and urinary Ca 2ϩ excretion (5).
The identification of germline heterozygous loss-and gainof-function mutations of G␣ 11 , which is encoded by the GNA11 gene on chromosome 19p13.3, that lead to forms of familial hypocalciuric hypercalcemia (FHH) or autosomal dominant hypocalcemia (ADH), respectively, has demonstrated the importance of this G-protein subunit in Ca 2ϩ o homeostasis (1,6,7). FHH is a genetically heterogeneous disorder that is inherited as an autosomal dominant condition, which is characterized by lifelong elevations of serum Ca 2ϩ concentrations in association with normal or mildly raised serum PTH levels and low urinary Ca 2ϩ excretion (8). FHH is considered to represent a benign disorder, however some patients may develop symptomatic hypercalcemia, pancreatitis, or chondrocalcinosis (8). FHH type 1 (FHH1, OMIM #145980) is caused by loss-of-function mutations of the CASR gene (9), and FHH type 2 (FHH2, OMIM #145981) is caused by loss-of-function G␣ 11 mutations, which comprise a L135Q missense substitution and in-frame isoleucine deletion at codon 199 or 200 (I199/200del) that impair CaSR signal transduction and were identified in two unrelated probands and families (1). ADH is also genetically heterogeneous and caused by germline gain-of-function mutations of the CASR and GNA11 genes, which lead to ADH types 1 (ADH1, OMIM #601198) and 2 (ADH2, OMIM #615361), respectively (1, 6 -10). Approximately 50% of ADH patients develop hypocalcemic symptoms such as paraesthesia, carpopedal spasms, and seizures, and Ͼ35% of patients harbor ectopic calcifications within the kidneys or basal ganglia (1,6,7,10). In contrast to germline gain-of-function G␣ 11 mutations, which affect Ca 2ϩ o homeostasis, somatic gain-of-function G␣ 11 mutations have been reported to lead to uveal melanoma, which is a primary intraocular tumor, by inducing constitutive up-regulation of proliferative signaling involving ERK, which is a component of the MAPK signaling pathway (11).
Compounds that selectively bind to the CaSR and allosterically modulate the function of this family C G-protein-coupled receptor (GPCR) represent a potential targeted therapy for patients with symptomatic forms of FHH and ADH. Indeed, cinacalcet, which is a licensed CaSR positive allosteric modulator, has been used effectively in FHH1 patients to manage symptomatic hypercalcemia and recurrent pancreatitis (12,13). Furthermore, negative allosteric CaSR modulators, known as calcilytics, have been demonstrated to ameliorate hypocalcemia in mouse models of ADH1 (14,15). The objective of this study was to undertake in vitro studies to determine whether allosteric modulators targeted to the CaSR may also rectify the loss-and gain-of-function associated with FHH2-and ADH2causing germline G␣ 11 mutations, respectively, and the up-regulation of ERK phosphorylation caused by a uveal melanomaassociated somatic G␣ 11 mutation. In addition, this study evaluated whether germline ADH2-causing gain-of-function G␣ 11 mutations may constitutively activate MAPK signaling and thus pose a risk for the development of uveal melanomas.
Studies involving siRNA knockdown of endogenous G␣ 11 were undertaken in HEK293 cells that stably expressed WT or mutant G␣ 11 proteins (HEK-G␣ 11 ). The HEK-G␣ 11 cells were generated using HEK293 T-Rex-Flp-in stable cell lines (Life Technologies), as reported (19). WT and mutant GNA11 constructs were cloned into the pcDNA5/FLP recombination target (FRT) expression vector (Life Technologies), and silent mutations introduced to render the constructs resistant to GNA11-targeted siRNA, thereby allowing investigation of the mutant G␣ 11 protein in the absence of endogenous WT G␣ 11 . GNA11 constructs were transiently transfected into T-Rex-Flp-in cells, and those cells expressing the G␣ 11 protein selected by culturing cells in medium containing Hygromycin (Gibco). The presence of the G␣ 11 protein and its resistance to siRNA was confirmed by Western blot analysis. Forty-eight hours prior to measuring Ca 2ϩ i responses, HEK-G␣ 11 cells were transiently transfected with the reported pEGFP-CaSR construct (9) and three different commercially available GNA11-targeted siRNA constructs (Trilencer-27 siRNA kit, catalogue number SR301839, Origene) or a commercially available scrambled siRNA (Trilencer-27 universal scrambled negative control siRNA duplex, catalogue number SR30004, Origene), and successful transfection confirmed by fluorescence microscopy, as described for pBI-CMV2expressing HEK-CaSR cells (1,16,17).
Measurement of Ca 2ϩ i Responses-The effect of allosteric CaSR modulators on cells expressing WT or mutant G␣ 11 proteins was assessed by a flow cytometry-based Ca 2ϩ i assay, as reported (1,16,17). In brief, 48 h after transfection, the cells were harvested, washed in Ca 2ϩ -and magnesium (Mg 2ϩ )-free Hank's balanced salt solution (HBSS) (Invitrogen), and loaded with 1 g/ml indo-1-acetoxymethylester (Indo-1-AM) (Molecular Probes) for 1 h at 37°C (1,16,17). Transfected cells were incubated with either a 20% aqueous solution of 2-hydroxypropyl-␤-cyclodextrin (Sigma) (vehicle), or positive or negative CaSR allosteric modulators, known as cinacalcet or NPS-2143, respectively, at concentrations ranging from 10 -40 nM for 1 h (15) (21). The baseline fluorescence ratio was measured for 2 min, the fluorescence ratio compared with the time was recorded, and data were collected for 2 min at each [Ca 2ϩ ] o , as described (1,16,17). Cytomation Summit software was used to determine the peak mean fluorescence ratio of the transient response after each individual stimulus, which was expressed as a percentage normalized response (1,16,17). Concentration-response curves were generated using a 4-parameter non-linear regression curve-fit model (GraphPad Prism) to calculate the halfmaximal (EC 50 ) and area under the curve (AUC) mean Ϯ S.E. responses for each separate experiment (17).
Measurement of ERK Phosphorylation-HEK-CaSR cells, transfected with WT or mutant G␣ 11 proteins for 24 h, were seeded in 48-well plates and cultured overnight in high glucose DMEM containing 10% FBS, prior to being incubated for 4 h with serum-free DMEM containing 0.5 mM Ca 2ϩ , 25 mM HEPES buffer with or without cinacalcet or NPS-2143 at 10 -500 nM concentrations. Cells were stimulated for 4 min with pre-warmed serum-free DMEM that contained Ca 2ϩ concentrations ranging from 0.5-10 mM, as reported (22), and lysed in Surefire lysis buffer. Alphascreen Surefire ERK phosphorylation assays were performed on whole cell lysates, as reported (23), and the fluorescence signal measured using a PHERAStar FS microplate reader (BMG Labtech) (23). ERK phosphorylation responses measured at each [Ca 2ϩ ] o were normalized to the mean responses of WT expressing cells and expressed as a fold-change of responses obtained at basal (0.5 Statistical Analysis-The Ca 2ϩ i and ERK phosphorylation responses of cells expressing WT or mutant G␣ 11 proteins were compared from a minimum of four experiments using the F-test and Mann-Whitney U test, respectively (1). All analyses were undertaken using GraphPad Prism (GraphPad), and are presented as mean Ϯ S.E. A value of p Ͻ 0.05 was considered significant for all analyses.

Effect of Cinacalcet on the Ca 2ϩ
i Responses of FHH2-associated G␣ 11 Mutations-The FHH2-associated L135Q and I199/ 200del G␣ 11 mutations have been reported to impair the sensitivity of CaSR-expressing cells to Ca 2ϩ o (1), and we hypothesized that cinacalcet-mediated allosteric activation of the CaSR would ameliorate the loss-of-function associated with germline mutations of G␣ 11 , thereby rectifying the signal transduction abnormalities in cells expressing these FHH2-associated mutant G␣ 11 proteins. To investigate this hypothesis, WT or mutant GNA11-pBI-CMV2 constructs were transiently transfected in HEK-CaSR cells and the effect of cinacalcet on the responses of Ca 2ϩ i concentrations ([Ca 2ϩ ] i ) to alterations in [Ca 2ϩ ] o was assessed. Expression of the CaSR and G␣ 11 was confirmed by fluorescence microscopy and/or Western blot analysis of whole-cell lysates (Fig. 1, A and B). CaSR expression, which was normalized by comparison to ␣-tubulin expression, did not differ between cells transfected with WT or FHH2associated mutant GNA11-pBI-CMV2 vectors when compared with cells transfected with empty vector, whereas the expression of G␣ 11 was greater in cells transfected with WT or mutant constructs (Fig. 1B). HEK-CaSR cells transiently transfected with WT or mutant G␣ 11 proteins were exposed to varying Indeed, 10 nM of cinacalcet induced a leftward shift of the mutant concentration-response curve, so that this was indistinguishable from that of WT-expressing cells (Fig. 1F). The addition of 10 and 20 nM cinacalcet lowered the EC 50 values of cells expressing the del199/200 G␣ 11 mutant (Fig. 1E). However, despite the del199/200 mutant having an almost identical EC 50 value to the Gln-135 G␣ 11 mutant protein, these cinacalcet doses were insufficient to rectify the loss-of-function associated with the del199/200 G␣ 11 mutant (Fig. 1E). Subsequently, when cinacalcet was added at a 40 nM concentration to cells expressing the del199/200 G␣ 11 mutant, this lowered the EC 50 value to 2.68 Ϯ 0.04 mM (Fig. 1E), so that the del199/200 mutant concentration-response curve overlapped with that of the WT G␣ 11 protein (Fig. 1G).
Effect of NPS-2143 on the Ca 2ϩ i Responses of ADH2-associated G␣ 11 Mutations-We previously reported the germline R181Q and F341L G␣ 11 mutations to enhance the sensitivity of CaSR-expressing cells to Ca 2ϩ o (1), thereby giving rise to the hypocalcemic disorder of ADH2. To determine whether allosteric inhibition of the CaSR can rectify the gain-of-function associated with ADH2-causing G␣ 11 mutations, WT or ADH2associated mutant GNA11-pBI-CMV2 vectors were transiently transfected into HEK-CaSR cells, and the responses of [Ca 2ϩ ] i to alterations in [Ca 2ϩ ] o assayed. Expression of the CaSR and G␣ 11 was demonstrated by fluorescence microscopy and/or Western blot analysis (Fig. 2, A and B). Western blot analysis confirmed an increase in the expression of G␣ 11 in cells transfected with WT or ADH2-associated mutant proteins, when compared with cells transfected with empty vector alone (Fig.  2B). An assessment of the Ca 2ϩ i responses of HEK-CaSR cells transiently transfected with WT or ADH2-associated mutant G␣ 11 proteins following stimulation with [Ca 2ϩ ] o , demonstrated cells expressing the Gln-181 or Leu-341 mutants to induce a leftward shift of the concentration-response curves (Fig. 2C) with a significant increase in AUC values and reduc-   (Fig. 2, D and E), as previously reported (1). The addition of NPS-2143 to cells expressing the Gln-181 G␣ 11 mutant revealed a 10 nM concentration of this calcilytic compound to nor-malize the mutant EC 50 value to 2.57 Ϯ 0.07 mM (Fig. 2E), so that the concentration-response curve resembled that of WT G␣ 11 (Fig. 2F), whereas 20 nM NPS-2143 significantly increased the mutant EC 50 value to 2.72 Ϯ 0.12 mM when compared with WT expressing cells (Fig. 2E). In contrast to these studies involving the Gln-181 G␣ 11 mutant protein, the addition of 20 nM NPS 2143 to cells expressing the Leu-341 G␣ 11 mutant did not significantly alter the EC 50 . (Fig. 2E). Indeed, NPS-2143 at a concentration of 30 nM was required to increase the Leu-341 mutant EC 50 value to 2.66 Ϯ 0.09 mM and rectify the shift in the mutant concentrationresponse curve (Fig. 2, E and G). 11 Protein-To determine whether CaSR-targeted drugs rectify the Ca 2ϩ i responses of FHH2-and ADH2-mutant expressing cells by directly influencing mutant G␣ 11 -signaling or by indirect effects on WT G␣ 11 protein that is endogenously expressed in HEK293 cells, siRNA knockdown of endogenous WT G␣ 11 was undertaken in HEK-G␣ 11 cells stably expressing WT, FHH2associated Gln-135, or ADH2-associated Gln-181 mutant G␣ 11 proteins. Western blot analysis demonstrated that siRNA with a scrambled sequence did not alter endogenous WT G␣ 11 expression in untransfected HEK293 cells (Fig. 3A). In contrast, GNA11-targeted siRNA reduced endogenous WT G␣ 11 expression in untransfected HEK293 cells (Fig. 3A), and decreased the level of transiently expressed WT G␣ 11 in HEK293 cells (Fig. 3B), but did not affect the levels of stably expressed WT or mutant G␣ 11 proteins in HEK-G␣ 11 cells (Fig.  3B), which contained constructs with silent mutations that had rendered them resistant to GNA11-targeted siRNA. CaSR constructs were transiently transfected into HEK-G␣ 11 cells, and CaSR expression confirmed by fluorescence microscopy (Fig.  3C). The effects of cinacalcet or NPS-2143 on the Ca 2ϩ i responses of the FHH2-and ADH2-associated G␣ 11 mutants were assessed following knockdown of endogenous WT G␣ 11 using GNA11-targeted siRNAs (Fig. 3, D-G). These studies revealed that: 10 nM of cinacalcet could rectify the rightward shift in the concentration-response curve and lower the significantly raised EC 50 of the FHH2-associated Gln-135 G␣ 11 mutant from a value of 3.85 Ϯ 0.12 mM to values of 3.23 Ϯ 0.1 mM and 3.17 Ϯ 0.08 mM, respectively, in the presence of GNA11-targeted or scrambled siRNA (Fig. 3, D and E), so that these values were not significantly different from HEK-G␣ 11 cells stably expressing WT G␣ 11 (EC 50 ϭ 3.33 Ϯ 0.06 mM); and that 10 nM of NPS-2143 could normalize the leftward shift of the concentration-response curve and increased the EC 50 of the ADH2-associated Gln-181 G␣ 11 mutant from a value of 2.70 Ϯ 0.07 mM to values of 3.26 Ϯ 0.06 mM and 3.11 Ϯ 0.08 mM, respectively, in the presence of GNA11-targeted or scrambled siRNA (Fig. 3, F and G), so that these values were not significantly different from WT-expressing HEK-G␣ 11 cells. Thus, these results show that CaSR-targeted drugs can influence the signaling responses of downstream mutant G␣ 11 proteins.

Effect of CaSR Allosteric Modulators on the Ca 2ϩ i Responses in Absence of Endogenously Expressed WT G␣
Effect of ADH2-associated G␣ 11 Mutants on MAPK Signaling-To investigate whether the germline R181Q and F341L ADH2-associated mutant G␣ 11 proteins may lead to constitutive up-regulation of MAPK signaling, WT and mutant GNA11-pBI-CMV2 vectors were transiently transfected into HEK-CaSR cells and fold-change ERK phosphorylation (phospho-ERK) responses assessed following exposure to varying [Ca 2ϩ ] o . The effects of the ADH2-associated mutants on phospho-ERK responses were compared with the uveal melanomaassociated Q209L G␣ 11 mutation (11). Following stimulation with Ca 2ϩ o , the germline Gln-181 and Leu-341 mutants were revealed to have significantly (p Ͻ 0.001) increased maximal phospho-ERK fold-change responses (Gln-181 ϭ 18.1 Ϯ 1.1, Leu-341 ϭ 18.3 Ϯ 0.9) compared with WT G␣ 11 (14.7 Ϯ 0.3), consistent with a gain-of-function (Fig. 4A). However, in the absence of Ca 2ϩ o stimulation, the basal phospho-ERK responses of the ADH2 mutants were demonstrated to not differ from WT G␣ 11 (Fig. 4, A and B), and thus these mutants are not constitutively activating. In contrast, the tumor-associated somatic Q209L G␣ 11 mutation led to both significantly (p Ͻ 0.0001) increased basal and maximal phospho-ERK foldchange responses when compared with the ADH2 mutants or WT G␣ 11 , consistent with a constitutive up-regulation of MAPK signaling (Fig. 4, A and B). The effect of NPS-2143 on the phospho-ERK responses of HEK-CaSR cells expressing the ADH2-associated Gln-181 or Leu-341 mutants, or the uveal melanoma-associated Leu-209 mutant, was also assessed. NPS-2143 was added at 10 and 30 nM concentrations to cells expressing the Gln-181 and Leu-341 mutants, respectively, as these doses had rectified the Ca 2ϩ i responses of the G␣ 11 mutants (Fig. 2, F and G). The addition of 10 and 30 nM NPS-2143 significantly lowered the maximal fold-change responses of the Gln-181 and Leu-341 mutants to 14.0 Ϯ 0.5 and 14.9 Ϯ 0.4, respectively, so that these values did not differ from the phospho-ERK responses of cells expressing WT G␣ 11 (Fig. 4, C and D). However, cells expressing the uveal melanoma-associated Leu-209 mutant required NPS-2143 at a higher dose of 500 nM to successfully rectify increases in phospho-ERK responses (Fig. 4E).

Discussion
Our studies demonstrate that cinacalcet and NPS-2143, which are allosteric CaSR activators and inactivators, respectively, can successfully rectify the loss-of-function associated with FHH2causing G␣ 11 mutations and the gain-of-function associated with G␣ 11 mutations that lead to ADH2 or uveal melanomas (1,11). Cinacalcet and NPS-2143 are allosteric modulators that are predicted to bind to the CaSR transmembrane domain (24) and influence receptor activity by altering its conformational status. These compounds have been reported to rectify the activity of FHH1and ADH1-associated mutant CaSR proteins in vitro (15,(25)(26)(27). However, the ability of these agents to normalize CaSR sensitivity in the presence of an abnormality downstream of the CaSR remained unknown. The in vitro findings of our study indicate allosteric modulation at the level of the receptor can rectify such loss-and gain-of-function associated with mutations of the intracellular G␣ 11 protein. Indeed, these studies demonstrate that pharmacological GPCR modulation may directly overcome abnormalities affecting the downstream effector G-protein rather than by indirect effects on endogenously expressed WT G-proteins.
However, the G␣ 11 mutations showed differences in their responsiveness to allosteric CaSR modulators. For example, our study of the FHH2 mutants revealed that a 4-fold increase in the cinacalcet dose was required to normalize the loss-of-function associated with I199/200del compared withthe L135Q mutation, despite both mutations having similar EC 50 values. Similarly, a 3-fold increase in the NPS-2143 dosage was required to rectify the gain-of-function due to the ADH2-associated F341L mutation Values are expressed as a percentage of the WT basal phospho-ERK response. The uveal melanoma-associated Leu-209 G␣ 11 mutant induces a significant phospho-ERK elevation when exposed to basal 0.5 mM [Ca 2ϩ ] o , whereas the basal phospho-ERK responses of the ADH2-associated G␣ 11 mutants are not significantly different compared with WT G␣ 11 . C-E, addition of NPS-2143 (2143) at 10, 30, and 500 nM concentrations significantly decreased the phospho-ERK responses of the ADH2-associated Gln-181 and Leu-341 G␣ 11 mutants, and the UV-associated Leu-209 mutant G␣ 11 protein, respectively, to values that were not significantly different from WT G␣ 11 . ***, p Ͻ 0.0001. when compared with the gain-of-function R181Q mutation, despite both mutations having similar EC 50 values. Thus, the I199/ 200del and F341L mutations showed diminished sensitivity to cinacalcet and NPS-2143, respectively, and these differences in the sensitivities of the mutants to CaSR-targeted drugs may be explained by a reported crystallography study, which showed residues homologous to Ile-199 and Phe-341, in the related G␣ s protein to be located at the interface between GPCR and G␣-subunit (28). Thus, G␣ 11 mutations located at the GPCR-G␣ interface may potentially influence the efficacy of CaSR allosteric modulators.
Cells expressing loss-and gain-of-function G␣ 11 mutants responded to nanomolar concentrations of cinacalcet (10 -40 nM, which is equivalent to 3.6 -14.3 ng/ml) and NPS-2143 (10 -30 nM, which is equivalent to 4.4 -13.3 ng/ml), respectively. However, previous in vitro studies of CaSR mutations leading to FHH and ADH have indicated that micromolar concentrations of these drugs may be required to rectify associated signal transduction abnormalities (25)(26)(27), and in vivo studies in WT rats have reported that the plasma drug concentrations of cinacalcet and NPS-2143 required to alter PTH secretion are Ն20 ng/ml and Ͼ100 ng/ml, respectively (29,30). The responsiveness of G␣ 11 mutants to low doses of CaSR-targeted drugs may be explained by the finding that these mutants induced only minor disturbances of CaSR signal transduction. Indeed, the FHH2 and ADH2 mutants were associated with up to a 30% shift in the EC 50 values of HEK-CaSR cells used in this study, whereas CaSR mutations leading to FHH1 and ADH1 generally cause a Ͼ50% shift in the EC 50 value (9,21,31,32). However, it remains to be established whether such low concentrations of calcimimetic and calcilytic drugs will be able to rectify in vivo the alterations in mineral homeostasis in FHH2 and ADH2 patients.
Somatic gain-of-function G␣ 11 mutations that induce constitutive MAPK activation have been reported in uveal melanoma and are associated with an increased likelihood of metastases (11). We therefore assessed the effects of germline ADH2associated R181Q and F341L gain-of-function G␣ 11 mutations on MAPK signaling by measuring phospho-ERK responses. Our studies demonstrated that the ADH2 G␣ 11 mutants induced a milder increase in ERK phosphorylation when compared with the uveal melanoma Q209L G␣ 11 mutant. Moreover, up-regulation of ERK phosphorylation by the ADH2-associated G␣ 11 mutants only occurred in the presence of Ca 2ϩ o stimulation, and therefore these R181Q and F341L G␣ 11 mutants do not harbor constitutive activity. These findings are consistent with a recent report of an ADH2associated R60L G␣ 11 mutation, which also enhanced MAPK activation in a non-constitutive manner (7). The finding that ADH2-associated mutations are not constitutively activating can be explained by their locations within the GTPase domain of the G␣ subunit. Thus, the Gln-209 residue, which is mutated in uveal melanomas (11), is required to spatially orientate the terminal phosphate group of G␣-bound GTP (33), thereby facilitating its hydrolysis and the conversion of GTP to GDP. Mutations affecting the Gln-209 residue have been shown to abolish GTP hydrolysis, thereby leaving the G␣ subunit in a permanent GTP-bound state of activation (34). In contrast, the Arg-181 and Phe-341 G␣ 11 residues , which are mutated in ADH2, are not located near to the terminal phosphate of GTP, and likely induce more indirect and subtle effects on GTP hydrolysis (1). The ADH2-associated G␣ 11 mutations represent the first reports of non-constitutively activating G-protein mutations (1,6,7), and the milder nature of these mutations is consistent with post-natal survival, in contrast to the constitutively activating Q209L mutation, which has been shown to be cytotoxic when expressed at high levels (35), and is likely to be embryonically lethal. The occurrence of non-constitutively activating G␣ 11 mutations that are tolerated in humans and heritable, highlights the potential for such germline mutations to affect other G-proteins and be associated with disease-related phenotypes, and this possibility remains to be explored.
In summary, our studies have revealed that germline gain-offunction G␣ 11 mutations induce non-constitutive alterations in MAPK signaling, and that CaSR-targeted compounds may rectify signaling disturbances caused by germline and somatic G␣ 11 mutations, which are associated with calcium disorders and tumorigenesis, respectively. These findings indicate that allosteric modulation at the level of the receptor may influence signaling disturbances associated with mutations of the downstream G-protein.