Impaired cotranslational processing of the calcium-sensing receptor due to signal peptide missense mutations in familial hypocalciuric hypercalcemia.

The CASR, a cell surface glycoprotein expressed in parathyroid gland and kidney, is critical for maintaining extracellular calcium homeostasis. The inherited disorders, familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT), are caused by inactivating mutations in the CASR gene. The CASR has an N-terminal, 19 amino acid signal peptide that is predicted to direct the nascent polypeptide chain, as it emerges from the ribosome, into the endoplasmic reticulum (ER). Here, we report the functional characterization of three CASR mutations identified in hypercalcemic/hyperparathyroid patients. The mutations, L11S, L13P and T14A, lie within the signal peptide hydrophobic core. When transiently transfected into kidney cells, L11S and L13P mutants demonstrated reduced intracellular and plasma membrane expression and signaling to the mitogen-activated protein kinase pathway in response to extracellular calcium relative to wild-type CASR and the T14A mutant. All mutant CASR RNAs translated into protein normally. In cotranslational processing assays, which test the functionality of the signal peptide in the early secretory pathway, the wild-type CASR and mutant T14A nascent polypeptides were targeted to microsomal vesicles, representing the ER, translocated into the vesicular lumen and underwent core N-glycosylation. In contrast, the L11S and L13P mutants failed to be inserted in the microsomes and undergo glycosylation. This is the first study examining the function of the CASR signal sequence and reveals that both L11S and L13P mutants are markedly impaired with respect to cotranslational processing, accounting for the observed parathyroid dysfunction.

[1]  J. Rastad,et al.  Familial hypercalcemia and hypercalciuria caused by a novel mutation in the cytoplasmic tail of the calcium receptor. , 2000, Journal of Clinical Endocrinology and Metabolism.

[2]  M. Ito,et al.  Possible involvement of inefficient cleavage of preprovasopressin by signal peptidase as a cause for familial central diabetes insipidus. , 1993, The Journal of clinical investigation.

[3]  P. Walter,et al.  Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. , 1994, Annual review of cell biology.

[4]  E. Brown,et al.  Familial Hypocalciuric Hypercalcemia Versus Typical Primary Hyperparathyroidism , 1978 .

[5]  M. Racchi,et al.  Human coagulation factor X deficiency caused by a mutant signal peptide that blocks cleavage by signal peptidase but not targeting and translocation to the endoplasmic reticulum. , 1993, The Journal of biological chemistry.

[6]  E. Brown,et al.  Familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Effects of mutant gene dosage on phenotype. , 1994, The Journal of clinical investigation.

[7]  J. Chudek,et al.  Functional characterization of calcium-sensing receptor codon 227 mutations presenting as either familial (benign) hypocalciuric hypercalcemia or neonatal hyperparathyroidism. , 2005, The Journal of clinical endocrinology and metabolism.

[8]  R. Lasker,et al.  An association between neonatal severe primary hyperparathyroidism and familial hypocalciuric hypercalcemia in three kindreds. , 1982, The New England journal of medicine.

[9]  E. Brown,et al.  Calcium-receptor-regulated parathyroid and renal function. , 1997, Bone.

[10]  E M Brown,et al.  Molecular Cloning and Functional Expression of Human Parathyroid Calcium Receptor cDNAs (*) , 1995, The Journal of Biological Chemistry.

[11]  H Baba,et al.  Mutation of the signal peptide-encoding region of the preproparathyroid hormone gene in familial isolated hypoparathyroidism. , 1990, The Journal of clinical investigation.

[12]  E. Brown,et al.  Markedly reduced activity of mutant calcium-sensing receptor with an inserted Alu element from a kindred with familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. , 1997, The Journal of clinical investigation.

[13]  G. Heijne A new method for predicting signal sequence cleavage sites. , 1986 .

[14]  E. Brown,et al.  Divalent cation metabolism. Familial hypocalciuric hypercalcemia versus typical primary hyperparathyroidism. , 1978, The American journal of medicine.

[15]  W. Simonds,et al.  Familial Isolated Hyperparathyroidism: Clinical and Genetic Characteristics of 36 Kindreds , 2002, Medicine.

[16]  M. A. Andersen,et al.  FAMILIAL BENIGN HYPERCALCAEMIA: HYPERCALCIURIA AND HYPOCALCIURIA IN AFFECTED MEMBERS OF A SMALL KINDRED , 1990, Clinical endocrinology.

[17]  A. Spiegel,et al.  Identification of the Sites of N-Linked Glycosylation on the Human Calcium Receptor and Assessment of Their Role in Cell Surface Expression and Signal Transduction* , 1998, The Journal of Biological Chemistry.

[18]  G. Blobel,et al.  Protein translocation across the endoplasmic reticulum. , 1994, Current opinion in cell biology.

[19]  S. Marx,et al.  Familial hypocalciuric hypercalcemia. Mild expression of the gene in heterozygotes and severe expression in homozygotes. , 1985, The American journal of medicine.

[20]  Samuel Refetoff,et al.  Partial deficiency of thyroxine-binding globulin-Allentown is due to a mutation in the signal peptide. , 2004, The Journal of clinical endocrinology and metabolism.

[21]  S. Brunak,et al.  Improved prediction of signal peptides: SignalP 3.0. , 2004, Journal of molecular biology.

[22]  G. Hendy,et al.  An acceptor splice site mutation in the calcium‐sensing receptor (CASR) gene in familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism , 2001, Human mutation.

[23]  A. Spiegel,et al.  Naturally occurring mutations of the extracellular Ca2+-sensing receptor: implications for its structure and function , 2003, Trends in Endocrinology & Metabolism.

[24]  M. Whyte,et al.  Calcium‐sensing receptor mutations in familial hypocalciuric hypercalcaemia with recurrent pancreatitis , 1996, Clinical endocrinology.

[25]  G. Hendy,et al.  CASRdb: calcium‐sensing receptor locus‐specific database for mutations causing familial (benign) hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemia , 2004, Human mutation.

[26]  R. Lasker,et al.  The Hypocalciuric or Benign Variant of Familial Hypercalcemia: Clinical and Biochemical Features in Fifteen Kindreds , 1981, Medicine.

[27]  Jurgen Seppen,et al.  A mutation which disrupts the hydrophobic core of the signal peptide of bilirubin UDP‐glucuronosyltransferase, an endoplasmic reticulum membrane protein, causes Crigler‐Najjar type IIs , 1996, FEBS letters.

[28]  E. Brown,et al.  Expression and Characterization of Inactivating and Activating Mutations in the Human Ca2+o-sensing Receptor* , 1996, The Journal of Biological Chemistry.

[29]  M. Hediger,et al.  Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid , 1993, Nature.

[30]  B. Teh,et al.  Genetic testing in familial isolated hyperparathyroidism: unexpected results and their implications , 2004, Journal of Medical Genetics.

[31]  M. Bai Structure-function relationship of the extracellular calcium-sensing receptor. , 2004, Cell calcium.

[32]  H. Arnqvist,et al.  Familial hypocalciuric hypercalcaemia: a study of four kindreds , 1989, Journal of internal medicine.

[33]  D. Damiani,et al.  Severe hypercalcemia in a 9-year-old Brazilian girl due to a novel inactivating mutation of the calcium-sensing receptor. , 2004, The Journal of clinical endocrinology and metabolism.

[34]  G. Hendy,et al.  Mutations of the calcium‐sensing receptor (CASR) in familial hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemia , 2000, Human mutation.

[35]  A. Spiegel,et al.  Maximal urine-concentrating ability: familial hypocalciuric hypercalcemia versus typical primary hyperparathyroidism. , 1981, The Journal of clinical endocrinology and metabolism.

[36]  J. Doppman,et al.  Familial hypocalciuric hypercalcemia: recognition among patients referred after unsuccessful parathyroid exploration. , 1980, Annals of internal medicine.

[37]  P. Adams,et al.  Familial hypocalciuric hypercalcaemia: evidence for continued enhanced renal tubular reabsorption of calcium following total parathyroidectomy. , 1984, Acta endocrinologica.

[38]  H Nielsen,et al.  Machine learning approaches for the prediction of signal peptides and other protein sorting signals. , 1999, Protein engineering.

[39]  G. Hendy,et al.  Recurrent familial hypocalcemia due to germline mosaicism for an activating mutation of the calcium-sensing receptor gene. , 2003, The Journal of clinical endocrinology and metabolism.

[40]  J. Seidman,et al.  Mutations in the human Ca2+-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism , 1993, Cell.

[41]  J. Seidman,et al.  Autosomal dominant hypocalcaemia caused by a Ca2+-sensing receptor gene mutation , 1994, Nature Genetics.

[42]  D. Heath Familial benign hypercalcemia , 1989, Trends in Endocrinology & Metabolism.

[43]  J. Shiloach,et al.  Expression, Purification, and Biochemical Characterization of the Amino-terminal Extracellular Domain of the Human Calcium Receptor* , 1999, The Journal of Biological Chemistry.

[44]  Martin R. Pollak,et al.  Familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism , 1995 .

[45]  H. Heath Familial benign (hypocalciuric) hypercalcemia. A troublesome mimic of mild primary hyperparathyroidism. , 1989, Endocrinology and metabolism clinics of North America.

[46]  Henry M. Kronenberg,et al.  Inefficient Membrane Targeting, Translocation, and Proteolytic Processing by Signal Peptidase of a Mutant Preproparathyroid Hormone Protein (*) , 1995, The Journal of Biological Chemistry.