Drugs that allosterically target the human calcium-sensing receptor (CaSR) have substantial therapeutic potential, but are currently limited. Given the absence of high-resolution structures of the CaSR, we combined mutagenesis with a novel analytical approach and molecular modeling to develop an "enriched" picture of structure-function requirements for interaction between Ca 2+ o and allosteric modulators within the CaSR's 7 transmembrane (7TM) domain. An extended cavity that accommodates multiple binding sites for structurally diverse ligands was identified. Phenylalkylamines bind to a site that overlaps with a putative Ca 2+ o -binding site and extends towards an extracellular vestibule. In contrast, the structurally and pharmacologically distinct AC-265347 binds deeper within the 7TM domains. Furthermore, distinct amino acid networks were found to mediate cooperativity by different modulators. These findings may facilitate the rational design of allosteric modulators with distinct and potentially pathway-biased pharmacological effects.

Cinacalcet is predominantly used to treat secondary hyperparathyroidism due to end-stage renal failure, but, more recently, its potential clinical efficacy in treating patients with loss-of-function mutations in the calcium-sensing receptor (CaSR) has been recognized. Many clinically relevant CaSR mutations are located in the heptahelical membrane spanning and extracellular loop regions of the receptor, where allosteric modulators are predicted to bind. The aim of the present study was to investigate the impact of such mutations on the pharmacoregulation of the CaSR by the positive and negative allosteric modulators, cinacalcet and NPS-2143, respectively. Both cinacalcet and NPS-2143 effectively rescued mutants whose cell surface expression was substantially impaired, suggesting that both classes of drug can stabilize a receptor conformation that is trafficked more effectively to the cell surface. In addition, functional impairments in almost all mutant CaSRs were rescued by either cinacalcet or NPS-2143 via restoration of intracellular signaling. There was a significantly greater ability of both compounds to modulate agonist-stimulated intracellular Ca(2+) mobilization than ERK1/2 phosphorylation, indicating that the allosteric modulators engender bias in agonist-stimulated CaSR signaling to different pathways. Three mutations (G(670)R, P(748)R, and L(773)R) altered the binding affinity of allosteric modulators to the CaSR, and 3 mutations (V(817)I, L(773)R, and E(767)K) altered the cooperativity between the allosteric modulator and Ca(2+)(o). These findings have important implications for the treatment of diseases associated with CaSR mutations using allosteric CaSR modulators and for analyzing the effects of mutations on the function and pharmacoregulation of the CaSR.

BACKGROUND AND PURPOSEClinical use of cinacalcet in hyperparathyroidism is complicated by its tendency to induce hypocalcaemia, arising partly from activation of calcium-sensing receptors (CaS receptors) in the thyroid and stimulation of calcitonin release. CaS receptor allosteric modulators that selectively bias signalling towards pathways that mediate desired effects [e.g. parathyroid hormone (PTH) suppression] rather than those mediating undesirable effects (e.g. elevated serum calcitonin), may offer better therapies. EXPERIMENTAL APPROACHWe characterized the ligand-biased profile of novel calcimimetics in HEK293 cells stably expressing human CaS receptors, by monitoring intracellular calcium (Ca 2+ i) mobilization, inositol phosphate (IP)1 accumulation, ERK1/2 phosphorylation (pERK1/2) and receptor expression. KEY RESULTSPhenylalkylamine calcimimetics were biased towards allosteric modulation of Ca 2+ i mobilization and IP1 accumulation. S,R-calcimimetic B was biased only towards IP1 accumulation. R,R-calcimimetic B and AC-265347 were biased towards IP1 accumulation and pERK1/2. Nor-calcimimetic B was unbiased. In contrast to phenylalkylamines and calcimimetic B analogues, AC-265347 did not promote trafficking of a loss-of-expression, naturally occurring, CaS receptor mutation (G 670 E). CONCLUSIONS AND IMPLICATIONSThe ability of R,R-calcimimetic B and AC-265347 to bias signalling towards pERK1/2 and IP1 accumulation may explain their suppression of PTH levels in vivo at concentrations that have no effect on serum calcitonin levels. The demonstration that AC-265347 promotes CaS receptor receptor signalling, but not trafficking reveals a novel profile of ligand-biased modulation at CaS receptors The identification of allosteric modulators that bias CaS receptor signalling towards distinct intracellular pathways provides an opportunity to develop desirable biased signalling profiles in vivo for mediating selective physiological responses. Abbreviations

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The soluble form of the chemokine fractalkine/CX 3 CL1 regulates microglia activation in the central nervous system (CNS), ultimately affecting neuronal survival. This study aims to determine whether CXCL12, another chemokine constitutively expressed in the CNS (known as stromal cell-derived factor 1; SDF-1), regulates cleavage of fractalkine from neurons. To this end, ELISA was used to measure protein levels of soluble fractalkine in the medium of rat neuronal cultures exposed to SDF-1. Gene arrays, quantitative RT-PCR, and Western blot were used to measure overall fractalkine expression in neurons. The data show that the rate of fractalkine shedding in healthy cultures positively correlates with in vitro differentiation and survival. In analogy to non-neuronal cells, metalloproteinases (ADAM10/17) are involved in cleavage of neuronal fractalkine as indicated by studies with pharmacologic inhibitors. Moreover, treatment of the neuronal cultures with SDF-1 stimulates expression of the inducible metalloproteinase ADAM17 and increases soluble fractalkine content in culture medium. The effect of SDF-1 is blocked by an inhibitor of both ADAM10 and -17, but only partially affected by a more specific inhibitor of ADAM10. In addition, SDF-1 also up-regulates expression of the fractalkine gene. Conversely, exposure of neurons to an excitotoxic stimulus (i.e. NMDA) inhibits ␣-secretase activity and markedly diminishes soluble fractalkine levels, leading to cell death. These results, along with previous findings on the neuroprotective role of both SDF-1 and fractalkine, suggest that this novel interaction between the two chemokines may contribute to in vivo regulation of neuronal survival by modulating microglial neurotoxic properties.