Molecular determinants of non-competitive antagonist binding to the mouse GPRC6A receptor

GPRC6A displays high sequence homology to the Ca²⁺-sensing receptor (CaSR). Here we report that the calcimimetic Calindol and the calcilytic NPS2143 antagonize increases in inositol phosphate elicited by l-ornithine-induced activation of mouse GPRC6A after transient coexpression with Gα_qG66D in HEK293 cells. The calcilytic Calhex 231 did not modulate this response. A three-dimensional model of the GPRC6A seven transmembrane domains (TMs) was constructed. It was used to identify seven residues strictly conserved within the CaSR and GPRC6A allosteric binding pockets, and previously demonstrated to interact with calcilytics or calcimimetics. The mutations F666A^3.32, F670A^3.36, W797A^6.48 caused a loss of l-ornithine ability to activate GPRC6A mutants. The F800A^6.51 mutant was not implicated in either Calindol or NPS 2143 recognition. The E816Q^7.39 mutation led to a loss of Calindol antagonist activity but was without effect on NPS2143 inhibitory response. In summary, these data suggest that Calindol is primarily anchored through an H-bond to E816^7.39 in TM7 and highlight important local differences at the level of the CaSR and GPRC6A allosteric binding pockets. We have identified the first antagonists of GPRC6A that could represent new tools to analyze GPRC6A functions and serve as chemical leads for the development of more specific modulators.     

Homology modeling of the transmembrane domain of the human calcium sensing receptor and localization of an allosteric binding site

A homology model for the human calcium sensing receptor (hCaR) transmembrane domain utilizing bovine rhodopsin (bRho) structural information was derived and tested by docking the allosteric antagonist, NPS 2143, followed by mutagenesis of predicted contact sites. Mutation of residues Phe-668 (helix II), Arg-680, or Phe-684 (helix III) to Ala (or Val or Leu) and Glu-837 (helix VII) to Ile (or Gln) reduced the inhibitory effects of NPS 2143 on [Ca2+]i responses. The calcimimetic NPS R-568 increases the potency of Ca2+ in functional assays of CaR. Mutations at Phe-668, Phe-684, or Glu-837 attenuated the effects of this compound, but mutations at Arg-680 had no effect. In all cases, mutant CaRs responded normally to Ca2+ or phenylalanine, which act at distinct site(s). Discrimination by the Arg-680 mutant is consistent with the structural differences between NPS 2143, which contains an alkyl bridge hydroxyl group, and NPS R-568, which does not. The homology model of the CaR transmembrane domain robustly accounts for binding of both an allosteric antagonist and agonist, which share a common site, and provides a basis for the development of more specific and/or potent allosteric modulators of CaR. These studies suggest that the bRho backbone can be used as a starting point for homology modeling of even distantly related G protein-coupled receptors and provide a rational framework for investigation of the contributions of the transmembrane domain to CaR function.     

Evidence for distinct cation and calcimimetic compound (NPS 568) recognition domains in the transmembrane regions of the human Ca2+ receptor

The Ca(2+) receptor, a member of the family 3 of G protein-coupled receptors (GPCR), responds not only to its primary physiological ligand Ca(2+) but also to other di- and trivalent metals (Mg(2+), Gd(3+)) and the organic polycations spermine and poly-l-Arginine. As has been found for other family 3 GPCRs, the large amino-terminal extracellular domain (ECD) of the Ca(2+) receptor is the primary Ca(2+) binding domain. To examine how the signal is propagated from the ECD to the seven-transmembrane core domain (7TM) we constructed a Ca(2+) receptor mutant (T903-Rhoc) lacking the entire ECD but containing the 7TM. We have found that this structure initiates signaling in human embryonic kidney (HEK) 293 cells stably expressing the construct. One or more cation recognition sites are also located within the 7TM. Not only Ca(2+), but also several other Ca(2+) receptor-specific agonists, Mg(2+), Gd(3+), spermine, and poly-l-Arginine, can activate T903-Rhoc truncated receptor-initiated phosphoinositide hydrolysis in HEK 293 cells. The phenylalkylamine compound, NPS 568, identified as a positive allosteric modulator of the Ca(2+) receptor can selectively potentiate the actions of Ca(2+) and other polycationic agonists on the T903-Rhoc receptor. Similarly, organic polycations synergistically activate T903-Rhoc with di- and trivalent metals. Alanine substitution of all the acidic residues in the second extracellular loop of the T903-Rhoc receptor significantly impairs activation by metal ions and organic polycations in the presence of NPS 568 but not the synergistic activation of Ca(2+) with poly-l-Arginine. These data indicate that although the ECD has been thought to be the main determinant for Ca(2+) recognition, the 7TM core of the Ca(2+) receptor contains activating site(s) recognizing Ca(2+) and Gd(3+) as well as the allosteric modulators NPS 568 and organic polycations that may play important roles in the regulation of receptor activation.     

L-phenylalanine and NPS R-467 synergistically potentiate the function of the extracellular calcium-sensing receptor through distinct sites

The extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) can be potentiated by allosteric activators including calcimimetics and l-amino acids. In this study, we found that many mutations had differential effects on the functional modulation of the CaR by these two allosteric activators, supporting the idea that these modulators act through distinct sites. 10 mm l-phenylalanine and 1 microm NPS R-467, submaximal doses of the two agents, each elicited similar modulation of R185Q. However, there are different relative potencies for these two modulators with some receptors being more responsive to l-phenylalanine and others being more responsive to NPS R-467. The responsiveness of the CaR to Ca(2+)(o) appears to be essential to observe the potentiating action of l-phenylalanine but not of NPS R-467 on the receptor. NPS R-467 reduces the Hill coefficients of the wild-type as well as mutant receptors, suggesting that engagement of all Ca(2+) binding sites is not required when the receptor is activated by NPS R-467. In contrast, l-phenylalanine has little effect on the Hill coefficients of mutant receptors. The two-site model is further supported by the observation that these two classes of modulators exert a synergistic effect on CaRs with inactivating mutations that are responsive to both modulators.     

Delineating a Ca2+ binding pocket within the venus flytrap module of the human calcium-sensing receptor

The Ca(2+)-sensing receptor (CaSR) belongs to the class III G-protein-coupled receptors (GPCRs), which include receptors for pheromones, amino acids, sweeteners, and the neurotransmitters glutamate and gamma-aminobutyric acid (GABA). These receptors are characterized by a long extracellular amino-terminal domain called a Venus flytrap module (VFTM) containing the ligand binding pocket. To elucidate the molecular determinants implicated in Ca(2+) recognition by the CaSR VFTM, we developed a homology model of the human CaSR VFTM from the x-ray structure of the metabotropic glutamate receptor type 1 (mGluR1), and a phylogenetic analysis of 14 class III GPCR VFTMs. We identified critical amino acids delineating a Ca(2+) binding pocket predicted to be adjacent to, but distinct from, a cavity reminiscent of the binding site described for amino acids in mGluRs, GABA-B receptor, and GPRC6a. Most interestingly, these Ca(2+)-contacting residues are well conserved within class III GPCR VFTMs. Our model was validated by mutational and functional analysis, including the characterization of activating and inactivating mutations affecting a single amino acid, Glu-297, located within the proposed Ca(2+) binding pocket of the CaSR and associated with autosomal dominant hypocalcemia and familial hypocalciuric hypercalcemia, respectively, genetic diseases characterized by perturbations in Ca(2+) homeostasis. Altogether, these data define a Ca(2+) binding pocket within the CaSR VFTM that may be conserved in several other class III GPCRs, thereby providing a molecular basis for extracellular Ca(2+) sensing by these receptors.     

Mutational analysis and molecular modeling of the allosteric binding site of a novel,selective, noncompetitive antagonist of the metabotropic glutamate 1 receptor

A model of the rmGlu1 seven-transmembrane domain complexed with a negative allosteric modulator, 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)- 1,6-dihydro-pyrimidine-5-carbonitrile (EM-TBPC) was constructed. Although the mGlu receptors belong to the family 3 G-protein-coupled receptors with a low primary sequence similarity to rhodopsin-like receptors, the high resolution crystal structure of rhodopsin was successfully applied as a template in this model and used to select residues for site-directed mutagenesis. Three mutations, F801(6.51)A, Y805(6.55)A, and T815(7.39)M caused complete loss of the [(3)H]EM-TBPC binding and blocked the EM-TBPC-mediated inhibition of glutamate-evoked G-protein-coupled inwardly rectifying K(+) channel current and [Ca(2+)](i) response. The mutation W798(6.48)F increased the binding affinity of antagonist by 10-fold and also resulted in a marked decrease in the IC(50) value (4 versus 128 nm) compared with wild type. The V757(5.47)L mutation led to a dramatic reduction in binding affinity by 13-fold and a large increase in the IC(50) value (1160 versus 128 nm). Two mutations, N7474(5.51)A and N7504(5.54)A, increased the efficacy of the EM-TBPC block of the glutamate-evoked [Ca(2+)](i) response. We observed a striking conservation in the position of critical residues. The residues Val-757(5.47), Trp-798(6.48), Phe-801(6.51), Tyr-805(6.55), and Thr-815(7.39) are critical determinants of the EM-TBPC-binding pocket of the mGlu1 receptor, validating the rhodopsin crystal structure as a template for the family 3 G-protein-coupled receptors. In our model, the aromatic ring of EM-TBPC might interact with the cluster of aromatic residues formed from Trp-798(6.48), Phe-801(6.51), and Tyr-805(6.55), thereby blocking the movement of the TM6 helix, which is crucial for receptor activation.     

Modeling and mutagenesis of the binding site of Calhex 231, a novel negative allosteric modulator of the extracellular Ca(2+)-sensing receptor

A model of the Ca2+-sensing receptor (CaSR) seven transmembrane domains was constructed based on the crystal structure of bovine rhodopsin. This model was used for docking (1S,2S,1'R)-N1-(4-chlorobenzoyl)-N2-[1-(1-naphthyl)ethyl]-1,2-diaminocyclohexane (Calhex 231), a novel potent negative allosteric modulator that blocks (IC50 = 0.39 microm) increases in [3H]inositol phosphates elicited by activating the human wild-type CaSR transiently expressed in HEK293 cells. In this model, Glu-8377.39 plays a pivotal role in anchoring the two nitrogen atoms of Calhex 231 and locating the aromatic moieties in two adjacent hydrophobic pockets delineated by transmembrane domains 3, 5, and 6 and transmembrane domains 1, 2, 3, and 7, respectively. To demonstrate its validity, we have mutated selected residues and analyzed the biochemical and pharmacological properties of the mutant receptors transfected in HEK293 cells. Two receptor mutations, F684A3.32 and E837A7.39, caused a loss of the ability of Calhex 231 to inhibit Ca2+-induced accumulation of [3H]inositol phosphates. Three other mutations, F688A3.36, W818A6.48, and I841A7.43, produced a marked increase in the IC50 of Calhex 231 for the Ca2+ response, whereas L776A5.42 and F821A6.51 led to a decrease in the IC50. Our data validate the proposed model for the allosteric interaction of Calhex 231 with the seven transmembrane domains of the CaSR. Interestingly, the residues at the same positions have been shown to delimit the antagonist-binding cavity of many diverse G-protein-coupled receptors. This study furthermore suggests that the crystal structure of bovine rhodopsin exhibits sufficient mimicry to the ground state of a very divergent class 3 receptor to predict the interaction of antagonists with the heptahelical bundle of diverse G-protein-coupled receptors.     

N2-benzyl-N1-(1-(1-naphthyl)ethyl)-3-phenylpropane-1,2-diamines and conformationally restrained indole analogues: development of calindol as a new calcimimetic acting at the calcium sensing receptor

The synthesis and calcimimetic activities of two new families of compounds are described. The most active derivatives of the first family, N(2)-(2-chloro-(or 4-fluoro-)benzyl)-N(1)-(1-(1-naphthyl)ethyl)-3-phenylpropane-1,2-diamine (4b and 4d, respectively, tested at 10 microM) produced 98+/-6% and 95+/-4%, respectively, of the maximal stimulation of [(3)H]inositol phosphates production obtained by 10mM Ca(2+) in CHO cells expressing the rat calcium sensing receptor (CaSR). The second family of calcimimetics was obtained by conformationally restraining the compounds of type 4 to provide the 2-aminomethyl derivatives 5. One of these compounds, (R)-2-[N-(1-(1-naphthyl)ethyl)aminomethyl]indole ((R)-5a, calindol), displayed improved calcimimetic activity compared to 4b and 4d as well as stereoselectivity. In the presence of 2mM Ca(2+), calindol stimulated [(3)H]inositol phosphates accumulation with an EC(50) of 1.0+/-0.1 or 0.31+/-0.05 microM in cells expressing the rat or the human CaSR, respectively. The calcimimetic activities of these novel compounds were shown to be due to a specific interaction with the CaSR.     

Calcium sensing receptor activation by a calcimimetic suggests a link between cooperativity and intracellular calcium oscillations

Activation of the calcium sensing receptor (CaR) by small increments in extracellular calcium (Ca(2+)(e)) induces intracellular calcium (Ca(2+)(i)) oscillations that are dependent on thapsigargin-sensitive intracellular calcium stores. Phenylalkylamines such as NPS R-568 are allosteric modulators (calcimimetics) that activate CaR by increasing the apparent affinity of the receptor for calcium. We determined, by fluorescence imaging with fura-2, whether the calcimimetic NPS R-568 could activate Ca(2+)(i) oscillations in HEK-293 cells expressing human CaR. NPS R-568 was more potent than Ca(2+)(e) at eliciting Ca(2+)(i) oscillations, particularly at low [Ca(2+)](e) (as low as 0.1 mm). The oscillation frequencies elicited by NPS R-568 varied over a 2-fold range from peak to peak intervals of 60-70 to 30-45 s, depending upon the concentrations of both Ca(2+)(e) and NPS R-568. Finally, NPS R-568 induced sustained (>15 min after drug removal) Ca(2+)(i) oscillations, suggesting slow release of the drug from its binding site. We exploited the potency of NPS R-568 for eliciting Ca(2+)(i) oscillations for structural studies. Truncation of the CaR carboxyl terminus from 1077 to 886 amino acids had no effect on the ability of Ca(2+) or NPS R-568 to induce Ca(2+)(i) oscillations, but further truncation (to 868 amino acids) eliminated both highly cooperative Ca(2+)-dependent activation and regular Ca(2+)(i) oscillations. Alanine scanning within the amino acid sequence from Arg(873) to His(879) reveals a linkage between the cooperativity for Ca(2+)-dependent activation and establishment and maintenance of intracellular Ca(2+) oscillations. The amino acid residues critical to both functions of CaR may contribute to interactions with either G proteins or between CaR monomers within the functional dimer.     

Calindol, a positive allosteric modulator of the human Ca(2+) receptor, activates an extracellular ligand-binding domain-deleted rhodopsin-like seven-transmembrane structure in the absence of Ca(2+)

The extracellular calcium-sensing human Ca(2+) receptor (hCaR),2 a member of the family-3 G-protein-coupled receptors (GPCR) possesses a large amino-terminal extracellular ligand-binding domain (ECD) in addition to a seven-transmembrane helical domain (7TMD) characteristic of all GPCRs. Two calcimimetic allosteric modulators, NPS R-568 and Calindol ((R)-2-{1-(1-naphthyl)ethyl-aminom-ethyl}indole), that bind the 7TMD of the hCaR have been reported to potentiate Ca(2+) activation without independently activating the wild type receptor. Because agonists activate rhodopsin-like family-1 GPCRs by binding within the 7TMD, we examined the ability of Calindol, a novel chemically distinct calcimimetic, to activate a Ca(2+) receptor construct (T903-Rhoc) in which the ECD and carboxyl-terminal tail have been deleted to produce a rhodopsin-like 7TMD. Here we report that although Calindol has little or no agonist activity in the absence of extracellular Ca(2+) for the ECD-containing wild type or carboxyl-terminal deleted receptors, it acts as a strong agonist of the T903-Rhoc. In addition, Ca(2+) alone displays little or no agonist activity for the hCaR 7TMD, but potentiates the activation by Calindol. We confirm that activation of Ca(2+) T903-Rhoc by Calindol truly the is independent using in vitro reconstitution with purified G(q). These findings demonstrate distinct allosteric linkages between Ca(2+) site(s) in the ECD and 7TMD and the 7TMD site(s) for calcimimetics.     

Improved model building and assessment of the Calcium-sensing receptor transmembrane domain

A three-dimensional model of the human Calcium-sensing receptor (CaSR) seven transmembrane domain was built via a novel sequence alignment method based on the conserved contacts in proteins using the crystal structure of bovine rhodopsin as the template. This model was tested by docking NPS 2143, the first identified allosteric antagonist of CaSR. In our model, Glu837 plays a critical role in anchoring the protonated nitrogen atom and hydroxy oxygen atom of NPS 2143. The phenyl moiety of the ligand contacts residues Phe668, Pro672, and Ile841. The naphthalene moiety is surrounded by several hydrophobic residues, including Phe684, Phe688, and Phe821. Our model appears to be consistent with all six residues that have been demonstrated to be critical for NPS 2143 binding, in contrast with existing homology models based on traditional sequence alignment of CaSR to rhodopsin. This provides validation of our sequence alignment method and the use of the rhodopsin backbone as the initial structure in homology modeling of other G protein-coupled receptors that are not members of the rhodopsin family.     

A region in the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+

Of 12 naturally occurring, activating mutations in the seven-transmembrane (7TM) domain of the human Ca2+ receptor (CaR) identified previously in subjects with autosomal dominant hypocalcemia (ADH), five appear at the junction of TM helices 6 and 7 between residue Ile819 and Glu837. After identifying a sixth activating mutation in this region, V836L, in an ADH patient, we studied the remaining residues in this region to determine whether they are potential sites for activating mutations. Alanine-scanning mutagenesis revealed five additional residues in this region that when substituted by alanine led to CaR activation. We also found that, whereas E837A did not activate the receptor, E837D and E837K mutations did. Thus, region Ile819-Glu837 of the 7TM domain represents a "hot spot" for naturally occurring, activating mutations of the receptor, and most of the residues in this region apparently maintain the 7TM domain in its inactive configuration. Unique among the residues in this region, Pro823, which is highly conserved in family 3 of the G protein-coupled receptors, when mutated to either alanine or glycine, despite good expression severely impaired CaR activation by Ca2+. Both the P823A mutation and NPS 2143, a negative allosteric modulator that acts on the 7TM through a critical interaction with Glu837, blocked activation of the CaR by various ADH mutations. These results suggest that the 7TM domain region Ile819-Glu837 plays a key role in CaR activation by Ca2+. The implications of our finding that NPS 2143 corrects the molecular defect of ADH mutations for treatment of this disease are also discussed.     

Amino Alcohol- (NPS-2143) and Quinazolinone-Derived Calcilytics (ATF936 and AXT914) Differentially Mitigate Excessive Signalling of Calcium-Sensing Receptor Mutants Causing Bartter Syndrome Type 5 and Autosomal Dominant Hypocalcemia

Introduction

Activating calcium sensing receptor (CaSR) mutations cause autosomal dominant hypocalcemia (ADH) characterized by low serum calcium, inappropriately low PTH and relative hypercalciuria. Four activating CaSR mutations cause additional renal wasting of sodium, chloride and other salts, a condition called Bartter syndrome (BS) type 5. Until today there is no specific medical treatment for BS type 5 and ADH. We investigated the effects of different allosteric CaSR antagonists (calcilytics) on activating CaSR mutants.

Methods

All 4 known mutations causing BS type 5 and five ADH mutations were expressed in HEK 293T cells and receptor signalling was studied by measurement of intracellular free calcium in response to extracellular calcium ([Ca²⁺]_o). To investigate the effect of calcilytics, cells were stimulated with 3 mM [Ca²⁺]_o in the presence or absence of NPS-2143, ATF936 or AXT914.

Results

All BS type 5 and ADH mutants showed enhanced signalling activity to [Ca²⁺]_o with left shifted dose response curves. In contrast to the amino alcohol NPS-2143, which was only partially effective, the quinazolinone calcilytics ATF936 and AXT914 significantly mitigated excessive cytosolic calcium signalling of all BS type 5 and ADH mutants studied. When these mutants were co-expressed with wild-type CaSR to approximate heterozygosity in patients, ATF936 and AXT914 were also effective on all mutants.

Conclusion

The calcilytics ATF936 and AXT914 are capable of attenuating enhanced cytosolic calcium signalling activity of CaSR mutations causing BS type 5 and ADH. Quinazolinone calcilytics might therefore offer a novel treatment option for patients with activating CaSR mutations.     

New insights into the pharmacology of the extracellular calcium sensing receptor

The extracellular calcium-sensing receptor (CaR) belongs to class III of G-protein coupled receptors. The CaR is expressed at the surface of the parathyroid cells and plays an essential role in the regulation of Ca2+ homeostasis through the control of parathyroid secretion. The CaR is activated by Ca2+ and Mg2+ present in the extracellular fluids, various di- and trivalent cations, L-aminoacids and charged molecules including several antibiotics. Calcimimetics potentiate the effect of Ca2+ and are proposed to be of therapeutic benefit for the treatment of both primary and secondary hyperparathyroidism. Calcilytics block the Ca2+-induced activation of the CaR. Three-dimensional models of the seven transmembrane domains of the human CaR have been used to identify specific residues implicated in the recognition of calcimimetics and calcilytics. These molecules should be useful for delineating the physiological roles played by the CaR in several tissues and for clarifying the direct effects attributed to extracellular Ca2+.     

A Type 1 Cholecystokinin Receptor Mutant That Mimics the Dysfunction Observed for Wild Type Receptor in a High Cholesterol Environment

Cholecystokinin (CCK) stimulates the type 1 CCK receptor (CCK1R) to elicit satiety after a meal. Agonists with this activity, although potentially useful for treatment of obesity, can also have side effects and toxicities of concern, making the development of an intrinsically inactive positive allosteric modulator quite attractive. Positive allosteric modulators also have the potential to correct the defective receptor-G protein coupling observed in the high membrane cholesterol environment described in metabolic syndrome. Current model systems to study CCK1R in such an environment are unstable and expensive to maintain. We now report that the Y140A mutation within a cholesterol-binding motif and the conserved, class A G protein-coupled receptor-specific (E/D)RY signature sequence results in ligand binding and activity characteristics similar to wild type CCK1R in a high cholesterol environment. This is true for natural CCK, as well as ligands with distinct chemistries and activity profiles. Additionally, the Y140A construct also behaved like CCK1R in high cholesterol in regard to its internalization, sensitivity to a nonhydrolyzable GTP analog, and anisotropy of a bound fluorescent CCK analog. Chimeric CCK1R/CCK2R constructs that systematically changed the residues in the allosteric ligand-binding pocket were studied in the presence of Y140A. This established increased importance of unique residues within TM3 and reduced the importance of TM2 for binding in the presence of this mutation, with the agonist trigger likely pulled away from its Leu³⁵⁶ target on TM7. The distinct conformation of this intramembranous pocket within Y140A CCK1R provides an opportunity to normalize this by using a small molecule allosteric ligand, thereby providing safe and effective correction of the coupling defect in metabolic syndrome.     

Calcium-sensing Receptor Biosynthesis Includes a Cotranslational Conformational Checkpoint and Endoplasmic Reticulum Retention

Metabolic labeling with [³⁵S]cysteine was used to characterize early events in CaSR biosynthesis. [³⁵S]CaSR is relatively stable (half-life ∼8 h), but maturation to the final glycosylated form is slow and incomplete. Incorporation of [³⁵S]cysteine is linear over 60 min, and the rate of [³⁵S]CaSR biosynthesis is significantly increased by the membrane-permeant allosteric agonist NPS R-568, which acts as a cotranslational pharmacochaperone. The [³⁵S]CaSR biosynthetic rate also varies as a function of conformational bias induced by loss- or gain-of-function mutations. In contrast, [³⁵S]CaSR maturation to the plasma membrane was not significantly altered by exposure to the pharmacochaperone NPS R-568, the allosteric agonist neomycin, or the orthosteric agonist Ca²⁺ (0.5 or 5 mm), suggesting that CaSR does not control its own release from the endoplasmic reticulum. A CaSR chimera containing the mGluR1α carboxyl terminus matures completely (half-time of ∼8 h) and without a lag period, as does the truncation mutant CaSRΔ868 (half-time of ∼16 h). CaSRΔ898 exhibits maturation comparable with full-length CaSR, suggesting that the CaSR carboxyl terminus between residues Thr⁸⁶⁸ and Arg⁸⁹⁸ limits maturation. Overall, these results suggest that CaSR is subject to cotranslational quality control, which includes a pharmacochaperone-sensitive conformational checkpoint. The CaSR carboxyl terminus is the chief determinant of intracellular retention of a significant fraction of total CaSR. Intracellular CaSR may reflect a rapidly mobilizable “storage form” of CaSR and/or may subserve distinct intracellular signaling roles that are sensitive to signaling-dependent changes in endoplasmic reticulum Ca²⁺ and/or glutathione.     

Clinical use of calcimimetics in the treatment of hyperparathyroidisms

Recognition of the role of the extracellular calcium sensing receptor (CaR) in mineral metabolism has greatly improved our understanding of calcium homeostasis. The activation of this receptor by small changes in the extracellular ionized calcium concentration (Ca(2+)ec) regulates parathormone (PTH) and calcitonin secretion, urinary calcium excretion and ultimately bone turnover. Cloning of CaR and discovery of mutations making the receptor less or more sensitive to calcium allowed a better understanding of several hereditary disorders characterized either by hyperparathyroidism or hypoparathyroidism. CaR became an ideal target for the development of compounds able to modulate the activity of CaR, activators (calcimimetics) as well as inhibitors (calcilytics). The calcimimetics are able to amplify the sensitivity of the CaR to Ca(2+)ec, suppressing PTH levels with a resultant fall in blood Ca2+. They dose-dependently reduce the secretion of PTH in vitro in cultured parathyroid cells, in animal models and in humans. In uremic animals, these compounds prevent parathyroid cell hyperplasia, normalize plasma PTH levels and bone remodelling. In uremic patients undergoing hemodialysis, the calcimimetics reduce plasma PTH concentration at short-term (12 weeks) as well as at long-term (2 years), serum calcium-phosphorus product and bone remodelling. After one year of treatment, these patients show a gain of bone mass of 2-3% at the femoral neck and at the total body. Contrarily, the calcilytics, by inhibiting CaR, can intermittently stimulate the secretion and the serum concentration of PTH. This results in an skeletal anabolic effect with a substantial increase in bone mineral density. They are potentially very interesting for the treatment of post-menopausal osteoporosis.     

Molecular Determinants of Allosteric Modulation at the M1 Muscarinic Acetylcholine Receptor

Benzylquinolone carboxylic acid (BQCA) is an unprecedented example of a selective positive allosteric modulator of acetylcholine at the M₁ muscarinic acetylcholine receptor (mAChR). To probe the structural basis underlying its selectivity, we utilized site-directed mutagenesis, analytical modeling, and molecular dynamics to delineate regions of the M₁ mAChR that govern modulator binding and transmission of cooperativity. We identified Tyr-85^2.64 in transmembrane domain 2 (TMII), Tyr-179 and Phe-182 in the second extracellular loop (ECL2), and Glu-397^7.32 and Trp-400^7.35 in TMVII as residues that contribute to the BQCA binding pocket at the M₁ mAChR, as well as to the transmission of cooperativity with the orthosteric agonist carbachol. As such, the BQCA binding pocket partially overlaps with the previously described “common” allosteric site in the extracellular vestibule of the M₁ mAChR, suggesting that its high subtype selectivity derives from either additional contacts outside this region or through a subtype-specific cooperativity mechanism. Mutation of amino acid residues that form the orthosteric binding pocket caused a loss of carbachol response that could be rescued by BQCA. Two of these residues (Leu-102^3.29 and Asp-105^3.32) were also identified as indirect contributors to the binding affinity of the modulator. This new insight into the structural basis of binding and function of BQCA can guide the design of new allosteric ligands with tailored pharmacological properties.     

Structural insights into activation of phosphatidylinositol 4-kinase (Pik1) by yeast frequenin (Frq1)

Yeast frequenin (Frq1), a small N-myristoylated EF-hand protein, activates phosphatidylinositol 4-kinase Pik1. The NMR structure of Ca2+-bound Frq1 complexed to an N-terminal Pik1 fragment (residues 121-174) was determined. The Frq1 main chain is similar to that in free Frq1 and related proteins in the same branch of the calmodulin superfamily. The myristoyl group and first eight residues of Frq1 are solvent-exposed, and Ca2+ binds the second, third, and fourth EF-hands, which associate to create a groove with two pockets. The Pik1 peptide forms two helices (125-135 and 156-169) connected by a 20-residue loop. Side chains in the Pik1 N-terminal helix (Val-127, Ala-128, Val-131, Leu-132, and Leu-135) interact with solvent-exposed residues in the Frq1 C-terminal pocket (Leu-101, Trp-103, Val-125, Leu-138, Ile-152, and Leu-155); side chains in the Pik1 C-terminal helix (Ala-157, Ala-159, Leu-160, Val-161, Met-165, and Met-167) contact solvent-exposed residues in the Frq1 N-terminal pocket (Trp-30, Phe-34, Phe-48, Ile-51, Tyr-52, Phe-55, Phe-85, and Leu-89). This defined complex confirms that residues in Pik1 pinpointed as necessary for Frq1 binding by site-directed mutagenesis are indeed sufficient for binding. Removal of the Pik1 N-terminal region (residues 8-760) from its catalytic domain (residues 792-1066) abolishes lipid kinase activity, inconsistent with Frq1 binding simply relieving an autoinhibitory constraint. Deletion of the lipid kinase unique motif (residues 35-110) also eliminates Pik1 activity. In the complex, binding of Ca2+-bound Frq1 forces the Pik1 chain into a U-turn. Frq1 may activate Pik1 by facilitating membrane targeting via the exposed N-myristoyl group and by imposing a structural transition that promotes association of the lipid kinase unique motif with the kinase domain.