MARKEDLY REDUCED ACTIVITY OF MUTANT CALCIUM-SENSING RECEPTOR WITH AN INSERTED ALU ELEMENT FROM A KINDRED WITH FAMILIAL HYPOCALCIURIC HYPERCALCEMIA AND NEONATAL SEVERE HYPERPARATHYROIDISM

Missense mutations have been identified in the coding region of the ex tracellular calcium-sensing receptor (CASR) gene and cause human autos omal dominant hypo- and hypercalcemic disorders. The functional effect s of several of these mutations have been characterized in either Xeno pus laevis oocytes or in human embryonic kidney (HEK293) cells. All of the mutations that have been examined to date, however, cause single putative amino acid substitutions. In this report, we studied a mutant CASR with an Alu-repetitive element inserted at codon 876, which was identified in affected members of families with the hypercalcemic diso rders, familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT), to understand how this insertion affects CASR function. After cloning of the Alu-repetitive element into the wi ld-type CASR cDNA, we transiently expressed the mutant receptor in HEK 293 cells. Expression of mutant and wild-type receptors was assessed b y Western analysis, and the effects of the mutation on extracellular c alcium (Ca-o(2+)) and gadolinium (Gd-o(3+)) elicited increases in the cytosolic calcium concentration (Ca-i(2+)) were examined in fura-2-loa ded cells using dual wavelength fluorimetry. The insertion resulted in truncated receptor species that had molecular masses some 30 kD less than that of the wild-type CASR and exhibited no Ca-i(2+) responses to either Ca-o(2+) or Gd-o(3+). A similar result was observed with a mut ated CASR truncated at residue 876. However, the Alu mutant receptor h ad no impact on the function of the coexpressed wild-type receptor. In terestingly, the Alu mutant receptor demonstrated decreased cell surfa ce expression relative to the wild-type receptor, whereas the CASR (A8 77stop) mutant exhibited increased cell surface expression. Thus, like the missense mutations that have been characterized to date in famili es with FHH, the Alu insertion in this family is a loss-of-function mu tation that produces hypercalcemia by reducing the number of normally functional CASRs on the surface of parathyroid and kidney cells. In vi tro transcription of exon 7 of the CASR containing the Alu sequence yi elded the full-length mutant product and an additional shorter product that was truncated due to stalling of the polymerase at the poly(T) t ract. In vitro translation of the mutant transcript yielded three trun cated protein products representing termination in all three reading f rames at stop codons within the Alu insertion. Thus sequences within t he Alu contribute to slippage or frameshift mutagenesis during transcr iption and/or translation.