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Naturally occurring mutations of the extracellular Ca21-sensing receptor: implications for its structure and function Jianxin Hu and Allen M. Spiegel Molecular Pathophysiology Section, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892, USA

The extracellular Ca21-sensing receptor is a member of the G-protein-coupled receptor family 3 in which agonists bind to a dimeric Venus-flytrap domain in the extracellular portion of the receptor. How agonist binding to this domain leads to activation of the seventransmembrane domain is a major unresolved question. Information derived from the three-dimensional structure of the Venus-flytrap domain of the related metabotropic glutamate type 1 receptor, and from naturally occurring mutations of the Ca21-sensing receptor identified in subjects with familial hypocalciuric hypercalcemia and autosomal dominant hypocalcemia offers new insights into the mechanism of receptor activation, and into the mechanism of action of allosteric modulators of the receptor. The cloning of the extracellular Ca2þ-sensing receptor (CaR) provided a new paradigm in signal transduction in which an extracellular ion, Ca2þ, serves as an agonist for a cell-surface receptor [1]. The CaR is expressed abundantly in parathyroid and kidney, where its activation inhibits parathyroid hormone (PTH) secretion and promotes urinary Ca2þ excretion, respectively [2]. The importance of the CaR in extracellular Ca2þ homeostasis is underscored by the identification of inactivating mutations in the CaR gene as the cause of familial hypocalciuric hypercalcemia (FHH) and neonatal severe primary hyperparathyroidism (NSPHT), and the identification of activating mutations as the cause of autosomal dominant hypocalcemia (ADH) [3]. The CaR is expressed in other tissues, where it might have roles beyond extracellular Ca2þ homeostasis (see [4] for a review). However, its central role in regulating PTH secretion has made it an attractive target for so-called calcimimetic and calcilytic drugs aimed at inhibiting or stimulating PTH secretion, respectively [5,6]. Notwithstanding its unique agonist, the CaR is a member of the G-protein-coupled receptor (GPCR) family 3 [7]. All GPCRs share the signature seven-transmembrane-spanning (7TM) domain. The assumption is that Corresponding author: J. Hu ([email protected]).

GPCR activation involves a conformational change of the membrane-spanning a helices, altering the disposition of intracellular loops, and thereby promoting activation of G proteins. For rhodopsin, a member of GPCR family 1, the three-dimensional (3D) structure of the receptor with its covalently bound ligand, retinal, has been solved, providing direct evidence for the interaction of ligand with specific residues of the membrane-spanning helices [8]. For members of GPCR family 3, which include, in addition to the CaR, multiple subtypes of metabotropic glutamate receptor (mGluR), the GABAB receptor and certain taste and pheromone receptors, evidence indicates that agonists bind to a dimeric, Venus-flytrap-like (VFT) domain within the large N-terminal extracellular domain (ECD) of the receptor. The VFT domain is linked to the 7TM domain by a cysteine-rich domain (Fig. 1a). Understanding how agonist binding to the VFT domain leads to receptor activation has important implications for designing drugs targeting family 3 GPCRs. Solution of the 3D structure of the VFT domain of the rat mGluR1 [9] offers important insights into agonistpromoted conformational changes, which are probably relevant for the CaR and other members of family 3. Here, we summarize the major features of the structure and function of the CaR, focusing in particular on what has been learned from the analysis of naturally occurring mutations. We also consider the implications of these results for understanding how allosteric modulators, such as type II calcimimetic drugs, might exert their effects. CaR structural and functional features The human CaR (hCaR) is a 1078-amino acid polypeptide comprising an N-terminal ECD, the 7TM domain and intracellular C-terminus (Fig. 2). The receptor couples primarily to members of the Gq/11 family to activate phosphoinositide breakdown [10]. The ECD contains 11 potential N-linked glycosylation sites (Fig. 2), of which at least three must be glycosylated for receptor expression at the cell surface [11]. Glycosylation does not appear to be crucial for CaR function, but rather for proper protein folding and trafficking. Biochemical characterization of a purified, secreted form of the ECD showed that the protein

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Fig. 1. The CaR is shown schematically in (a) its inactive (free) form and (b) its active (agonist-bound) form. Protomers of the CaR dimer are colored blue and red, respectively. The VFT domain (LB1 and LB2) and cysteine-rich domain (Cys-rich) of one protomer are labeled, and loop 2 with its two intermolecular disulfide bonds linking each LB1 protomer is shown. The 7TM domain is shown with its three extracellular loops (top) and its three intracellular loops and C terminus (bottom) connecting seven membrane-spanning a helices (cylinders). Agonist binding [not shown in (b)] to a cleft between LB1 and LB2 leads to VFT closure and a rotation about the dimer interface (note the change in loop 2 configuration). Abbreviations: CaR, Ca2þ-sensing receptor; LB1, lobe 1; LB2, lobe 2; 7TM, seven-transmembrane; VFT, Venus-flytrap.

begins with tyrosine 20, consistent with cleavage of a hydrophobic signal peptide (Fig. 2). Tryptic proteolysis of the purified protein revealed two major cleavage sites between residues 360 and 380 and between 520 and 570 [12]. These presumptively surface-exposed cleavage sites correspond to loop 3 and the junction of the VFT and cysteine-rich domains, respectively (Fig. 2). VFT domain Determination of the 3D structure of residues 33– 522 of the rat mGluR1 (equivalent to 20 – 540 of the hCaR) showed that this portion of the ECD is a bilobed VFT, structurally similar to bacterial periplasmic binding proteins [9]. A model of the VFT domain of the hCaR (Fig. 3) based on the mGluR1 crystal structure shows that each lobe comprises a helices and b sheets connected by short loops, with the N-terminal lobe (LB1) containing four longer loops, designated loops 1 – 4 in Fig. 2. Studies of mutant CaRs with deletions of parts of each of these loops revealed that a large part (365 –385) of loop 3 could be deleted without impairing receptor function, but that deletions of loop 1 (50 – 59) or loop 4 (438 – 445), although not impairing receptor expression, reduced CaR activation [13]. Deletions of loop 2, which is disordered in the mGluR1 crystal structure, increased Ca2þ sensitivity of the mutant CaR, similar to the results seen in a random mutagenesis study of loop 2 [14]. Agonist binding Ca2þ activates the CaR at mM concentrations, implying a much lower affinity Ca2þ-binding site than for intracellular Ca2þ-binding proteins, such as calmodulin. Activation of the CaR shows positive cooperativity, raising the http://tem.trends.com

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possibility of multiple Ca2þ-binding sites within the VFT or elsewhere, but other explanations are also possible [10]. In addition to Ca2þ, other polyvalent cations such as Gd3þ and polycationic compounds, such as spermine and neomycin, act as agonists for the CaR. This has led to the suggestion that polycationic agonists might activate the CaR by screening negatively charged residues, abundant throughout the ECD, but particularly seen within the C-terminal lobe (LB2) residues 215– 251 (Fig. 2). The crystal structure of the glutamate-bound form of the mGluR1 VFT revealed the key residues in LB1 and LB2 involved in agonist binding [9]. Studies of chimeric receptors [15 –17] show that the predominant agonistbinding site for the CaR, and probably most other family 3 GPCRs, resides within the VFT domain. However, the specific amino acids responsible for Ca2þ binding to the CaR have yet to be defined. Of the 13 residues shown to be involved in glutamate binding to the mGluR1 VFT, six are identical or conservatively substituted in the hCaR [LB1: Ser165 (mGluR1) ¼ Ser147 (hCaR), Thr188 , Ser170; LB2: Asp208 ¼ Asp190, Tyr236 ¼ Tyr218, Gly293 ¼ Gly273, Asp318 , Glu297]. Three of these residues, Ser147, Ser170 [16] and Asp190 [18], when artificially mutated to alanine, impair CaR activation. L -Amino acids allosterically enhance CaR sensitivity to Ca2þ, and studies of the Ser170Ala mutant suggest that the amino acidbinding site is related to that for Ca2þ itself [19]. Dimerization of the VFT Immunoblots of the intact CaR and of the soluble secreted ECD performed under reducing and non-reducing conditions indicate that the CaR is a dimer linked by intermolecular disulfide bond(s) within the ECD. Mutagenesis of the ten cysteines within the VFT (Fig. 2) indicated that cysteines 60, 101, 236, 358, 395, 437 and 449 are crucial for normal receptor expression and function, whereas cysteines 129, 131 and 482 could be mutated without loss of function [20]. The mGluR1 crystal structure shows that the equivalent pairs of cysteines to CaR 60 – 101, 358– 395 and 437 –449 form three intramolecular disulfides, which are probably crucial for the structural integrity of the VFT. The crucial importance of Cys236, the equivalent of which in mGluR1 is located free in LB2, is unexplained. Cys482 is not conserved even within vertebrate CaRs, consistent with lack of a crucial function. Although mutation of either Cys129 or Cys131 in loop 2 did not cause loss of function, the double mutant is expressed largely as a monomer under denaturing, non-reducing conditions, identifying these as the residues that are crucial for CaR dimerization. mGluRs also homodimerize through an intermolecular disulfide involving a conserved cysteine in loop 2 (residue 140 in the rat mGluR1). Studies with the soluble rat mGluR1 VFT containing a Cys140Ala mutation show that non-covalent interactions, in addition to an intermolecular disulfide, are involved in dimerization [21]. The crystal structure of the mGluR1 VFT shows an extensive dimer interface involving residues in the N-terminal portion of LB1 and within the proximal portion of LB2 [22]. A mutant mGluR1 with alanine substituted for isoleucine 120, a key residue within the dimer interface,

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Q A V F S L T A E L A K S V T N C T D F I R Y G L T L N P L L A P S S N I E E I A F I M A Q L WR F G R F N Y R I N K R62M R66C LOOP 2 120 I T138M L159P S170A G143E S147A T151M D 140 160 S L N L D E F C N C S E H I P S T I A V V G A T G S G V S T A V A N L L G L F Y I P Q V S Y A S S S R L L S N K N Q 180 F N124K L174R N178D K E127A/K C129F/S C131W S137P Y161C L125P Y218S D215G P221L E228Q E191K F128L S 220 200 240 F ES F D I C I D R E E A E E R F K E I G P RG Y D D D A A I T G VWNWR F Y E I I D AMA T A QHE D N P I T R L L P221S R220W/Q R185Q Q245R E250K R227L/Q D190A I 260 280 300 S Q Y S D E E E I Q H V V E V I QN S T A K V I V V F S S G P D L E P L I K E I V R R N I T G K I WL A S E AW A S S S E297K 340 320 360 L I E Q L H CN F T E EWF E K A F GN H V S K R P H V K K L F E R F G P I QG A K L A F G I T GG V VH F Y Q PMA G LOOP 3 A 420 380 K G P L P V D T F L RGH E E S GD R F S N S S T A F R P L C T GD E N I S S V E T P Y I D Y T H L R I S Y N VY LA 400 V LOOP 4 C395R Y 480 460 440 S I G C E D F T V Q E GMN N T F N L H R L H K L V QWA E V K K I D A C S G N T F L G R G P L C T Y I D Q L A H A D End of VFT L 540 500 520 V G N Y S I I NWH L S P E DG S I V F K E V G Y Y N V Y A K K G E R L F I N E E K I LWS G F S RE V P F S N C S R G553R G549R D C582Y F589L 560 600 580 C L I E K A I C S T H N E N SWF D D P C K NC A S A D T E D S Y E GD P C E V C E F CC T P E GE I I G K R T G A E F Start of Rho-C-hCaR L E604K S F832S W NQ E L E D E I I F I R680C Y R A835T T T Q681H S C C T W R E K F V S E837A D L S H G F612S Q A R P P E P748R Q Y P V PA Exo-loop 2 S G E Exo-loop 1 F T Exo-loop 3 E P G I L MA T Y L G I V I A I S A G F A L Y L773R A F L G670E/R S L I F L P A A824S V I W S A A L S F T I L FG I F S S A F C I V S820F F G L S G Y T V817I V L GI F I A843E L C C L V W MQ I C A L L L C C L I V I L A T F I F S F I F F I C A I C T C S A F F F N V S657Y S M L F806S F788C V L I F F V F L Y I K L L L V G L F A F T I I I L K T N L L F Y S L F I F F F E K K R Q L K K 863 Cyto-loop 3 Cyto-loop 2 Cyto-loop 1 V A S R F L P N E R R N L N L S K L P E N F N T G V R A R795W T P I V K F E A K I P T S F H R K WW N E799K T I E End of Rho-C-hCaR S895-V1075 in-frame deletion F881L E 920 900 880 V A T S CR PQ P F P D E S N S K S S I S S S P T S G T S GG L S S S R KR S V N S R R L T A R A A V K F A HA T E 903 R 960 980 940 Q K Q Q Q P L A L T QQ EQ QQQ P L T L P Q Q Q R S QQQ P R C K Q K V I F G SG T V T F S L S F D E PQ K N A M A 1000 1040 1020 H Q L G T EQ V T L D L D T E G C Q L P L L PQ H R T L T D S S K Q A E L S NQ H T S N G EV E P R QD GG V P G D P 1078 1060 E E L S P A L V V S S S Q S F V I S G G G S T V T E N V V N S COOH

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Fig. 2. The amino acid sequence of the hCaR (single letter code): cysteines (black), N-linked glycosylation sites (?), signal peptide, beginning and end of VFT domain, loops 1 –4 of the VFT domain LB1, and the start and end of the ECD-deleted Rho-C –hCaR construct are highlighted. Inactivating mutations causing FHH/NSPHT are shown in red (three artificial mutations involving the putative Ca2þ-binding site are boxed), and activating mutations causing ADH are shown in green. E837A, highlighted in blue, is an artificial mutation that revealed the involvement of the glutamate residue in binding the calcimimetic drug, NPS R-568. Abbreviations: ADH, autosomal dominant hypocalcemia; ECD, extracellular domain; FHH, familial hypocalciuric hypercalcemia; hCaR, human Ca2þ-sensing receptor; NSPHT, neonatal severe primary hyperparathyroidism; Rho-C, rhodopsin N-terminal residues 1– 20 added and C-terminal residues 904 –1078 of hCaR deleted.

abolished receptor activation, indicating the crucial role of the dimer interface in this process [23]. Dimerization of the CaR also involves non-covalent interactions, in addition to the two intermolecular disulfide bonds [24,25]. The functional importance of CaR dimerization is demonstrated by complementation of function between CaR monomers, each with distinct mutations in different domains of the receptor. Mutants involving the VFT or the intracellular C-terminus each http://tem.trends.com

show minimal function when expressed as homodimers, but recover some function when coexpressed to form heterodimers [26]. However, no complementation was seen when a VFT mutant was coexpressed with either a mutant truncated in the first intracellular loop or a mutant with the cysteine-rich domain deleted, suggesting that functional complementation requires two intact cysteine-rich and 7TM domains for communication between the VFT and 7TM domains [18].

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Fig. 3. A model of the three-dimensional structure of the CaR VFT dimer: a helices in red, b sheets in yellow, loops and turns in purple. Inter- and intramolecular disulfides are not shown. Lobes 1 and 2 (LB1 and LB2, respectively), loop 2, and each protomer are labeled. Loop 2 is depicted arbitrarily in the model because this loop was unstructured in the mGluR1 VFT crystal structure. The interface between lobes 1 and 2 is the putative agonist-binding site. The dimer interface runs along the vertical axis between the two protomers. Naturally occurring activating mutations are highlighted in green, and are primarily located along the dimer interface in loop 2 (top) and between the LB2 protomers (bottom). Abbreviations: CaR, Ca2þ-sensing receptor; mGluR, metabotropic glutamate receptor; VFT, Venus-flytrap.

Comparison of the glutamate-bound, ‘active’ versus antagonist-bound, ‘inactive’ structures of the mGluR1 VFT revealed several important differences [9,22]: (1) the VFT is closed in the glutamate-bound and open in the antagonist-bound structures; (2) residues equivalent to hCaR 117 – 123 in loop 2 form an ordered extension of an a helix of LB1 in the inactive form, but are disordered, along with the remainder of loop 2, in the active form; (3) agonistpromoted VFT closure leads to a 708 rotation of one monomer relative to the other about an axis perpendicular to the dimer interface; and (4) VFT closure-promoted rotation of the monomers permits LB2 domains to move ˚ closer than in the open VFT conformation, where 26 A electrostatic repulsion keeps them further apart. Apposition of the LB2 domains in the agonist-bound state might cause concomitant movement of the cysteine-rich domains linked to LB2. These changes in the CaR are shown in Fig. 1. Cysteine-rich domain The VFT and 7TM domains are linked by an 84-residue region containing nine closely spaced cysteines (Fig. 2), termed the cysteine-rich domain. With the exception of the GABAB receptor, which lacks this domain, other family 3 GPCRs contain the same nine cysteines with conserved spacing. Mutation of any of these cysteines to serine severely impairs the expression and function of the CaR [20]. Although chimeric hCaRs, in which the mGluR1 cysteine-rich domain is substituted for that of the hCaR, preserve some degree of function, deletion of the cysteinerich domain abolishes CaR activation, in spite of the http://tem.trends.com

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preservation of some cell-surface expression [17]. This suggests that the cysteine-rich domain plays a key role in signal transmission between the VFT and 7TM domains. The region between the end of the VFT, residue 528, and the initial Cys542 of the cysteine-rich domain appears relatively permissive of modifications, because neither insertion of an additional ten residues in a splice variant of the hCaR nor introduction of a hexapeptide TEV (tobacco etch virus) protease recognition site following Glu536 impairs function [27]. Analysis of the products of TEV protease cleavage at a site artificially inserted between the VFT and cysteine-rich domains demonstrated that the dimeric CaR VFT domain is not linked by disulfide bonds to either the cysteine-rich or 7TM domains. 7TM domain and intracellular C-terminus A CaR truncation mutant lacking the entire ECD was expressed in Xenopus oocytes and shown to retain some responsiveness to agonists such as Gd3þ [15]. A similar truncation mutant with N-terminal residues 1 –20 of bovine rhodopsin fused to hCaR Ala600 (Fig. 2; Rho-C – hCaR) shows excellent cell-surface expression and is activated by Ca2þ when added with an allosteric modulator, NPS R-568 [28]. These results suggest that the 7TM domain, in addition to the VFT, might contain sites for polycation binding and CaR activation. Mutagenesis of the acidic residues in extracellular loops 1 – 3 does not abolish Ca2þ activation of the receptor. In fact, mutation of acidic residues 758, 759 or 767 enhances Ca2þ sensitivity of the receptor, suggesting that these residues in extracellular loop 2 might help maintain the CaR in its inactive state. Lack of constitutive activity of Rho-C – hCaR contrasts with constitutively activated ECD-deletion mutants of the thyrotropin receptor. The 7TM of this last receptor might be directly inhibited by its ECD [29]. Much of the 216 residue C-terminus of the receptor (residues 889– 1078) can be truncated without impairing cell-surface expression and activation [30]. Nonetheless, the C-terminus might be responsible for other properties of the CaR, such as its positively cooperative response to Ca2þ [31], and its binding to a scaffold protein, filamin-A [32]. Inactivating mutations in FHH and NSPHT Inactivating mutations of the CaR cause a right-shift in set point for Ca2þ inhibition of PTH secretion and for stimulation of urinary Ca2þ excretion, leading to relative hypercalcemia and hypocalciuria in subjects with FHH and NSPHT. The severity of alteration in the biochemical phenotype correlates with the type of mutation. Null mutations that prevent CaR expression cause mild FHH when heterozygous, but cause NSPHT when homozygous or compound heterozygous. Heterozygous mutations that permit CaR expression but impair function might cause more severe FHH or NSPHT by acting as dominant negatives of the wild-type CaR, presumably through heterodimerization. Truncation of the hCaR proximal to residue 888 disrupts receptor function; thus, frameshift and nonsense mutations causing such truncation (not shown in Fig. 2) are inactivating mutations (e.g. Arg648stop produces an inactive CaR with a single transmembrane

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domain [33]). Missense mutations causing FHH/NSPHT might inactivate the CaR by impairing normal folding and cell-surface expression or by preventing Ca2þ activation of the properly expressed receptor. Over 30 inactivating missense mutations in FHH/NSPHT have been identified to date, and their distribution is nonrandom (Fig. 2). More than half cluster between residues 13 and 297 of the ECD, whereas only one has been reported between residues 298 and 548 [34,35]. A homozygous Leu13Pro mutation within the signal peptide identified in a nine-year-old girl with severe hypercalcemia presumptively impairs normal CaR expressionp. Three mutations, Glu297Lys, Tyr218Ser and Arg66Cys, involve residues equivalent to those participating in glutamate binding in mGluR1. These mutations might directly impair Ca2þ binding; however, Arg66Cys might inactivate by interfering with formation of the crucial 60 – 101 disulfide. Similarly, Cys395Arg prevents formation of the crucial 358– 395 disulfide. Given the important role of agonist-promoted rotation about the dimer interface demonstrated for mGluR1, it is not surprising that many inactivating CaR mutations involve residues at the dimer interface of LB1 (Ser53Pro, Pro55Leu, Leu159Pro, Asn178Asp and possibly Tyr161Cys and Leu174Arg) and LB2 (Asp215Gly, Arg220Trp/Gln, Pro221Ser and Arg227Leu/Gln). We speculate that such mutations impair the crucial agonist-promoted rotation about the dimer interface, thus blocking CaR activation. This might also be true of Arg185Gln located between part of the LB1 dimer interface and Asp190, a residue that is probably involved in Ca2þ binding. Interestingly, both Arg185Gln and Arg227Leu, identified as heterozygous mutations in NSPHT, behave as dominant negatives. Three mutations located just after loop 2, Ser137Pro, Thr138Met and Gly143Glu, might cause inactivation by preventing the agonist-dependent conformational change within that loop. Three mutations within the cysteine-rich domain, Gly549Arg, Gly553Arg and Cys582Tyr, probably impair normal folding of this domain. Of the missense mutations in the 7TM domain, Arg680Cys in extracellular loop 1 has been suggested to impair formation of a putative disulfide linking Cys677 and Cys765, and Arg795Trp in intracellular loop 3 might impair G-protein coupling. Val817Ile might impair an activation-dependent conformational change within TM6. Activating mutations in ADH Heterozygous, activating mutations in subjects with ADH cause a left-shift in the Ca2þ set point, leading to relative hypocalcemia and hypercalciuria. A Bartter syndromelike phenotype has also been reported in some subjects with ADH [36,37]. With the exception of an in-frame deletion, Ser895-Val1075 [38], activating mutations in ADH are missense mutations. Such mutations presumably * Miyashiro, K. et al. (2002) Severe hypercalcemia in a 9-year-old Brazilian girl due to a novel inactivating mutation (L13P) of the calcium sensing receptor. The 24th Annual Meeting of the American Society for Bone and Mineral Research, September 20 –24, 2002. San Antonio, Texas, USA (Abstract M439). http://tem.trends.com

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act by relieving inhibitory constraints that maintain the CaR in its inactive conformation. Most ADH mutations increase CaR sensitivity to Ca2þ, rather than causing constitutive activation. As with naturally occurring inactivating mutations, ADH mutations are clustered in particular regions of the CaR (Fig. 2). Most occur at the presumptive dimer interfaces of LB1 (Thr151Met, loop 2 residues Ala116Thr, Asn118Lys, Asn124Lys, Leu125Pro, Glu127Ala/Lys, Phe128Leu, Cys129Phe/Ser and Cys131Trp) and of LB2 (Pro221Leu, Glu228Gln and Gln245Arg) (Fig. 3). We speculate that these mutations enhance Ca2þ sensitivity by facilitating agonist-promoted dimer rotation. Note that Pro221Ser, the FHH counterpart of Pro221Leu, inactivates the CaR, suggesting that not only mutation of proline, but the particular substitution, defines the functional consequence. CaR activation by Glu191Lys might reflect the effect of the charge difference on the adjacent Asp190, presumptively involved in Ca2þ binding. Phe589Leu, Glu604Lys and Phe612Ser are located between the end of the cysteine-rich domain and the first transmembrane domain. Given the probable role of this crucial linker in communication between the VFT and 7TM domains, we speculate that these mutations act by facilitating communication between agonist-bound VFT and 7TM domains. Within the 7TM domain, a cluster of mutations in transmembrane helices 5, 6 and 7, and extracellular loop 3 (Fig. 2) suggests that movement of these helices relative to each other could be a crucial event in CaR activation. Indeed, Ala843Glu causes constitutive activation, whether expressed in the full-length CaR or the ECD-deleted Rho-C – hCaR [39]. Glu799Lys in intracellular loop 3 might be the counterpart to the inactivating mutation Arg795Trp, suggesting that increased positive charge in this loop enhances G-protein coupling. Allosteric modulators The phenylalkylamines, so-called type II calcimimetics NPS R-467 and NPS R-568, act as positive allosteric modulators of the CaR, enhancing its sensitivity to Ca2þ without activating it by themselves [5]. They are selective for the CaR, failing to modulate closely related GPCRs, such as mGluR1. They inhibit PTH secretion, and might prove useful in the treatment of primary and secondary hyperparathyroidism [40]. NPS R-568 acts on the 7TM domain of the CaR; alanine substitution for Glu837, at the top of TM7, nearly abolishes the response to NPS R-568 without affecting CaR expression or response to Ca2þ [28]. NPS R-467 restores some function to inactivating FHH mutations such as Arg185Gln [41]. Allosteric modulators, both positive and negative, have been identified for other family 3 GPCRs [42], and these also bind to the 7TM domain. In spite of limited sequence identity between rhodopsin and mGluR1, a model of the mGluR1 7TM domain proved instructive in defining the binding pocket of a negative allosteric modulator [43]. Trp798, Phe801 and Tyr805 in TM6, and Thr815 at the top of TM7 proved crucial for modulator binding. The authors suggested that the drug prevents agonist-induced motion of TM6, shown to occur upon rhodopsin activation. The same TM6 residues are conserved in hCaR (Trp818,

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Phe821 and Tyr825), and Glu837 might be equivalent to mGluR1 Thr815. We speculate that sequence differences in extracellular loop 3 and elsewhere in the 7TM domain might contribute to differential specificity of mGluR1 and CaR allosteric modulators. Furthermore, subtle differences in configuration of drugs binding to the 7TM domain might either prevent (negative modulators) or promote (positive modulators) the helical motion required for receptor activation. Activating CaR mutations in the 7TM domain might share a common mechanism of action with type II calcimimetics. Negative modulators of the CaR, so-called calcilytic drugs such as NPS 2143, block Ca2þ activation of the CaR, thereby stimulating PTH secretion, an effect that could prove useful in the treatment of osteoporosis [6]. The site and mechanism of action of such drugs has not been reported, and it would be interesting to test whether calcilytics suppress CaR-activating ADH mutations. Conclusions and future directions Studies of naturally occurring activating and inactivating CaR mutations, comparison with the 3D mGluR1 VFT structures, and studies with allosteric modulators enable us to formulate a model for CaR activation (Fig. 1). Ca2þ binding to the VFT promotes closure of LB2 upon LB1, followed by rotation about the dimer interface. This conformational change in the VFT is transmitted to the 7TM domains by rotation of the cysteine-rich domains, leading to movement of the TM helices within and possibly between 7TM domains. The questions left by this model point to important future research directions: † Determination of all residues responsible for Ca2þ binding; a direct agonist-binding assay would be useful. † Definition of loop 2 structure in the active and inactive states of the receptor. † Determination of the disulfide connectivity and 3D structure of the cysteine-rich domain; how does the structure change upon VFT closure? † Determination of the 3D structure of the 7TM domain in its activated and inactive states; definition of the binding pocket of allosteric modulators. † Definition of the interactions between agonist-bound VFT, cysteine-rich and 7TM domains; do they make direct contact or does VFT closure exert an indirect effect on the 7TM domains via the cysteine-rich domains? Addressing these questions should enable us to make more informed designs of drugs targeting the CaR, ultimately leading to improved treatment of disorders of Ca2þ metabolism. Acknowledgements We are grateful to P.K. Goldsmith, P.J. Steinbach and K.A. Jacobson, and to our former colleagues at the NIH who participated in several studies described in this review.

References 1 Brown, E.M. et al. (1993) Cloning and characterization of an extracellular Ca(2 þ )-sensing receptor from bovine parathyroid. Nature 366, 575– 580 2 Brown, E.M. and MacLeod, R.J. (2001) Extracellular calcium sensing and extracellular calcium signaling. Physiol. Rev. 81, 239 – 297 http://tem.trends.com

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