PMID- 9109436
OWN - NLM
STAT- MEDLINE
DCOM- 19970509
LR  - 20181113
IS  - 0021-9738 (Print)
IS  - 0021-9738 (Linking)
VI  - 99
IP  - 8
DP  - 1997 Apr 15
TI  - Markedly reduced activity of mutant calcium-sensing receptor with an inserted Alu 
      element from a kindred with familial hypocalciuric hypercalcemia and neonatal 
      severe hyperparathyroidism.
PG  - 1917-25
AB  - Missense mutations have been identified in the coding region of the extracellular 
      calcium-sensing receptor (CASR) gene and cause human autosomal dominant hypo- and 
      hypercalcemic disorders. The functional effects of several of these mutations 
      have been characterized in either Xenopus 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 disorders, 
      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 wild-type CASR 
      cDNA, we transiently expressed the mutant receptor in HEK293 cells. Expression of 
      mutant and wild-type receptors was assessed by Western analysis, and the effects 
      of the mutation on extracellular calcium (Ca2+(o)) and gadolinium (Gd3+(o)) 
      elicited increases in the cytosolic calcium concentration (Ca2+(i)) were examined 
      in fura-2-loaded 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 Ca2+(i) responses to either Ca2+(o) or 
      Gd3+(o). A similar result was observed with a mutated CASR truncated at residue 
      876. However, the Alu mutant receptor had no impact on the function of the 
      coexpressed wild-type receptor. Interestingly, the Alu mutant receptor 
      demonstrated decreased cell surface expression relative to the wild-type 
      receptor, whereas the CASR (A877stop) mutant exhibited increased cell surface 
      expression. Thus, like the missense mutations that have been characterized to 
      date in families with FHH, the Alu insertion in this family is a loss-of-function 
      mutation that produces hypercalcemia by reducing the number of normally 
      functional CASRs on the surface of parathyroid and kidney cells. In vitro 
      transcription of exon 7 of the CASR containing the Alu sequence yielded the 
      full-length mutant product and an additional shorter product that was truncated 
      due to stalling of the polymerase at the poly(T) tract. In vitro translation of 
      the mutant transcript yielded three truncated protein products representing 
      termination in all three reading frames at stop codons within the Alu insertion. 
      Thus sequences within the Alu contribute to slippage or frameshift mutagenesis 
      during transcription and/or translation.
FAU - Bai, M
AU  - Bai M
AD  - Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 
      Boston, Massachusetts 02115, USA.
FAU - Janicic, N
AU  - Janicic N
FAU - Trivedi, S
AU  - Trivedi S
FAU - Quinn, S J
AU  - Quinn SJ
FAU - Cole, D E
AU  - Cole DE
FAU - Brown, E M
AU  - Brown EM
FAU - Hendy, G N
AU  - Hendy GN
LA  - eng
GR  - DK09436/DK/NIDDK NIH HHS/United States
GR  - DK41415/DK/NIDDK NIH HHS/United States
GR  - DK44588/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Clin Invest
JT  - The Journal of clinical investigation
JID - 7802877
RN  - 0 (DNA Primers)
RN  - 0 (DNA, Complementary)
RN  - 0 (Receptors, Calcium-Sensing)
RN  - 0 (Receptors, Cell Surface)
RN  - 11089-65-9 (Tunicamycin)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Animals
MH  - Base Sequence
MH  - Calcium/blood/*urine
MH  - Cell Line
MH  - DNA Primers/genetics
MH  - DNA, Complementary/genetics
MH  - Genes, Dominant
MH  - Glycosylation
MH  - Humans
MH  - Hypercalcemia/*genetics/*metabolism
MH  - Hyperparathyroidism/*genetics/*metabolism
MH  - Immunohistochemistry
MH  - Infant, Newborn
MH  - Mutation
MH  - Receptors, Calcium-Sensing
MH  - Receptors, Cell Surface/chemistry/*genetics/*metabolism
MH  - Repetitive Sequences, Nucleic Acid
MH  - Transfection
MH  - Tunicamycin/pharmacology
PMC - PMC508016
EDAT- 1997/04/15 00:00
MHDA- 1997/04/15 00:01
PMCR- 1997/04/15
CRDT- 1997/04/15 00:00
PHST- 1997/04/15 00:00 [pubmed]
PHST- 1997/04/15 00:01 [medline]
PHST- 1997/04/15 00:00 [entrez]
PHST- 1997/04/15 00:00 [pmc-release]
AID - 10.1172/JCI119359 [doi]
PST - ppublish
SO  - J Clin Invest. 1997 Apr 15;99(8):1917-25. doi: 10.1172/JCI119359.