The calcium-sensing receptor in human disease : molecular genetics and structure-function analysis

Inherited disorders of calcium homeostasis, familial hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT) are caused by inactivating mutations, and autosomal dominant hypocalcemia (ADH) is caused by activating mutations in the calcium-sensing receptor (CASR). This receptor belongs to group C of the G-protein-coupled receptor (GPCR) superfamily, one of the largest groups of cell-surface receptors. The CASR is expressed abundantly in the parathyroid gland and in the cells lining the kidney tubule and senses small changes in circulating calcium concentration and once activated it inhibits parathyroid hormone (PTH) secretion and renal tubule calcium reabsorption. The CASR undergoes core (immature) N-linked glycosylation in the endoplasmic reticulum (ER). Once appropriately folded, the core-glycosylated receptor transits through the Golgi apparatus, becomes fully (mature) glycosylated, and is expressed at the cell surface. The main focus of this Ph.D thesis work was a functional characterization of mutant CASRs. At present over 100 CASR mutations have been published. Our lab has created a database http://www.casrdb.mcgill.ca/, of these mutations which is regularly updated. The CASRdb helps to better understand and analyze the mutations with respect to clinical and biochemical profiles, and aids in receptor structure-function analyses in addition to providing an important communication link between researchers working on this receptor and related areas. We examined the DNAs from patients with calcium homeostasis disorders and identified mutations and polymorphisms in the coding region of the CASR gene in several of these individuals. Most of the mutations are novel and some are recurring. Two of the identified mutations (L11S and T14A) are in the signal peptide of the receptor (another mutant, L13P, was previously reported but not analyzed functionally). In the CASR, the N-terminal signal peptide of 19 amino acids likely plays a key role in directing the nascent polypeptide chain into the ER. We demonstrated that indeed the wild-type CASR, and mutant T14A, nascent polypeptides were targeted to the ER in vitro, and underwent core N-glycosylation. In contrast, the L11S and L13P mutants did not translocate into the ER and did not undergo glycosylation. This study is the first to examine the function of the CASR signal sequence and shows that naturally occurring mutations that disrupt the hydrophobic core of this region lead to impaired cotranslational processing causing parathyroid dysfunction. It is the first study to analyze the impairment of a GPCR's signal peptide region resulting in human disease. The CASR exists at the plasma membrane as a homodimer although it is unclear at which point in the biosynthetic pathway dimerization occurs. To address this issue, we have analyzed wild-type and mutant CASRs harboring R66H or R66C inactivating mutations (identified in FHH patients), which cause the receptor to be retained in the ER. Using a biochemical and biophysical analysis we demonstrated that the wild-type, R66H and R66C mutants were dimerized in the ER whereas another mutant, N583X, was not. Hence, constitutive CASR dimerization occurs in the ER and is likely to be necessary, but is not sufficient, for exit of the receptor from the ER and trafficking to the cell surface. FHH may present atypically as familial isolated hyperparathyroidism (FIHP). I have performed in vitro analyses of mutations L159P, E250K, V268delfsX273, T445A, and R886P identified in FIHP kindreds. In addition, we have identified two novel inactivating mutations (M74L and L521delfs.X555) in FHH patients. We have analyzed all mutants by transiently transfecting them in human embryonic kidney (HEK293) cells and comparing them to the wild-type receptor. Western blot analysis demonstrated that the mutant receptors were, in general, as well expressed as the wild-type receptor. However, we observed that the 2 truncation mutants (V268delfsX273 and L521delfsX555) migrated at lower molecular weights. Immunocytochemistry and confocal microscopy studies showed that both of these mutants, as well as some of the others, had impaired cell surface expression. Several mutants had impaired MAPK responses to increasing extracellular calcium concentrations relative to the wild-type receptor. On the other hand, T445A behaved like the wild-type receptor in all the assays, suggesting that this change represented a rare benign polymorphism. In addition, three common CASR polymorphisms (A986S, R990G and Q1011E) were identified in several individuals, and were expressed at similar levels to wild-type on western blot analysis. R886P was the least impaired mutant in all assays. E250K was responsive to some extent in the MAPK assay, but the rest of the mutants had clearly impaired function. Identification and functional analyses of naturally-occurring CASR mutations provides important clinical information to physicians and patients. In addition, assessment of functional deficiencies of particular mutants provides new insight into how the receptor works.

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