Class C GPCRs

CaR Recently, the potential for pharmacological chaperone activity has been demonstrated in vitro for the CaR, which belongs to the Class C GPCR family and contains a large calcium-binding ECD. The CaR plays a critical role in calcium homeostasis, translating changes in plasma calcium levels into reciprocal changes in parathyroid hormone secretion from the parathyroid glands. Inactivating mutations in CaR produce a benign form of hypercalcemia when present in the heterozygous state, termed familial hypocalciuric hypercalcemia (FHH), while homozygous mutations produce a much more severe hypercal-cemic disorder resulting from marked hyperparathyroidism, called neonatal severe hyperparathyroidism (NSHPT) (for review, see Reference 223). These diseases are characterized by generalized resistance to extracellular calcium and increased circulating parathyroid hormone levels. Since the first report describing inactivating mutations in the CaR [224] - more than 225 missense mutations have been identified, with at least 30 resulting in a loss-of-function phenotype ( A majority of the mutations occur in the ECD, with others found in TM domains II and VI, extracellular loops I and II, intracellular loop III, and the carboxy-terminal tail of the receptor. A number of these mutations have been shown to result in severe ER retention, including R66C, R680C, R795W, and G549R, while others showed partial ER retention, including R185Q and V817I (for review, see References 180, 193).

A series of allosteric modulators (agonist and antagonist) were recently tested against the wild-type CaR, as well as against receptors with activating and inactivating mutations [193]. By Western blot analysis, increased levels of mature wild - -ype receptor and four inactive mutants (R185Q in the ECD, R680C in TM domain III, R795W in intracellular loop III, and V817I in TM domain VI) were seen after incubation with the allosteric agonist NPS - 568. Similarly, increased plasma membrane localization of the wild-type and mutant receptors was also observed. Importantly, functional rescue was proportional to the level of plasma membrane expression. In contrast, the allosteric antagonist NPS-2143 showed no effect against these mutant forms with inactivating mutations and decreased the activity of these mutants. This is in contrast to what has been observed for the V2R and GnRHR, for which antagonists were shown to act as pharmacological chaperones (described above). Also, for the 8-opioid receptor, both agonists and antagonists were found to have pharmacological chaperone properties [225]. Whether this difference results from the allosteric nature of the compounds tested for the CaR or is a characteristic of the receptor itself is unclear.

It should be noted that in clinical studies, NPS-568 effectively lowered circulating levels of parathyroid hormone in dialysis patients with secondary hyperparathyroidism [226], suggesting that long-term treatment with calcimi-metics can upregulate the levels of wild-type CaR, thus providing possibilities for intervention in diseases affecting calcium homeostasis.

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