Mutational analysis of the carboxyl-terminal region of the human calcium-sensing receptor

K. Ray, G. F. Fan, P. K. Goldsmith, Allen M. Spiegel

Research output: Contribution to journalArticle

Abstract

The importance of the cytoplasmic regions of the Calcium-sensing receptor (CAR) for cell surface expression and signal transduction was analysed. Two truncation mutants terminating at residues 706 and 802 within the second and third intracellular loops, .respectively, and four progressively shorter carboxyterminal truncation mutants terminating at residues 865, 874,888, 907, were transfected into HEK-293 cells. CaR expression was measured by immunoblotting and cell surface expression by an intact cell immuno-peroxidase-labeling technique. Signal transduction was assessed by measuring dose-dependent stimulation of phosphoinositide hydrolysis by calcium. Results indicate that all seven transmembrane helices are necessary for proper folding and processing of the hCaR as judged by lack of cell surface expression of T706 and T806 mutants. T888 and T907 mutants with 25 and 40 residues, respectively, of carboxy-terminal tail showed identical expression and signal transduction as the wild type receptor. T865 and T874 mutants, with 2 and 11 amino acid long carboxy-terminal tails,respectively, showed a reduction in cell surface expression and no phosphoinositide response to extracellular calcium. However, reduced stimulation of phosphoinositol accumulation was far more dramatic than the corresponding reduction in receptor expression. We conclude that the membrane-proximal portion ofthe hCaR carboxy-terminal tail plays a key role in normal expression of the receptor and may include amino acid residues importemt for G protein (Gq/11) activation.

Original languageEnglish (US)
JournalFASEB Journal
Volume11
Issue number9
StatePublished - 1997
Externally publishedYes

Fingerprint

Calcium-Sensing Receptors
Signal transduction
calcium
receptors
Tail
mutants
Phosphatidylinositols
Signal Transduction
signal transduction
tail
Gq-G11 GTP-Binding Protein alpha Subunits
cells
Calcium
Amino Acids
Peroxidase
HEK293 Cells
Hydrolysis
labeling techniques
amino acids
Immunoblotting

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Mutational analysis of the carboxyl-terminal region of the human calcium-sensing receptor. / Ray, K.; Fan, G. F.; Goldsmith, P. K.; Spiegel, Allen M.

In: FASEB Journal, Vol. 11, No. 9, 1997.

Research output: Contribution to journalArticle

@article{bcc3252035b04cc38ade68cb415e89d8,
title = "Mutational analysis of the carboxyl-terminal region of the human calcium-sensing receptor",
abstract = "The importance of the cytoplasmic regions of the Calcium-sensing receptor (CAR) for cell surface expression and signal transduction was analysed. Two truncation mutants terminating at residues 706 and 802 within the second and third intracellular loops, .respectively, and four progressively shorter carboxyterminal truncation mutants terminating at residues 865, 874,888, 907, were transfected into HEK-293 cells. CaR expression was measured by immunoblotting and cell surface expression by an intact cell immuno-peroxidase-labeling technique. Signal transduction was assessed by measuring dose-dependent stimulation of phosphoinositide hydrolysis by calcium. Results indicate that all seven transmembrane helices are necessary for proper folding and processing of the hCaR as judged by lack of cell surface expression of T706 and T806 mutants. T888 and T907 mutants with 25 and 40 residues, respectively, of carboxy-terminal tail showed identical expression and signal transduction as the wild type receptor. T865 and T874 mutants, with 2 and 11 amino acid long carboxy-terminal tails,respectively, showed a reduction in cell surface expression and no phosphoinositide response to extracellular calcium. However, reduced stimulation of phosphoinositol accumulation was far more dramatic than the corresponding reduction in receptor expression. We conclude that the membrane-proximal portion ofthe hCaR carboxy-terminal tail plays a key role in normal expression of the receptor and may include amino acid residues importemt for G protein (Gq/11) activation.",
author = "K. Ray and Fan, {G. F.} and Goldsmith, {P. K.} and Spiegel, {Allen M.}",
year = "1997",
language = "English (US)",
volume = "11",
journal = "FASEB Journal",
issn = "0892-6638",
publisher = "FASEB",
number = "9",

}

TY - JOUR

T1 - Mutational analysis of the carboxyl-terminal region of the human calcium-sensing receptor

AU - Ray, K.

AU - Fan, G. F.

AU - Goldsmith, P. K.

AU - Spiegel, Allen M.

PY - 1997

Y1 - 1997

N2 - The importance of the cytoplasmic regions of the Calcium-sensing receptor (CAR) for cell surface expression and signal transduction was analysed. Two truncation mutants terminating at residues 706 and 802 within the second and third intracellular loops, .respectively, and four progressively shorter carboxyterminal truncation mutants terminating at residues 865, 874,888, 907, were transfected into HEK-293 cells. CaR expression was measured by immunoblotting and cell surface expression by an intact cell immuno-peroxidase-labeling technique. Signal transduction was assessed by measuring dose-dependent stimulation of phosphoinositide hydrolysis by calcium. Results indicate that all seven transmembrane helices are necessary for proper folding and processing of the hCaR as judged by lack of cell surface expression of T706 and T806 mutants. T888 and T907 mutants with 25 and 40 residues, respectively, of carboxy-terminal tail showed identical expression and signal transduction as the wild type receptor. T865 and T874 mutants, with 2 and 11 amino acid long carboxy-terminal tails,respectively, showed a reduction in cell surface expression and no phosphoinositide response to extracellular calcium. However, reduced stimulation of phosphoinositol accumulation was far more dramatic than the corresponding reduction in receptor expression. We conclude that the membrane-proximal portion ofthe hCaR carboxy-terminal tail plays a key role in normal expression of the receptor and may include amino acid residues importemt for G protein (Gq/11) activation.

AB - The importance of the cytoplasmic regions of the Calcium-sensing receptor (CAR) for cell surface expression and signal transduction was analysed. Two truncation mutants terminating at residues 706 and 802 within the second and third intracellular loops, .respectively, and four progressively shorter carboxyterminal truncation mutants terminating at residues 865, 874,888, 907, were transfected into HEK-293 cells. CaR expression was measured by immunoblotting and cell surface expression by an intact cell immuno-peroxidase-labeling technique. Signal transduction was assessed by measuring dose-dependent stimulation of phosphoinositide hydrolysis by calcium. Results indicate that all seven transmembrane helices are necessary for proper folding and processing of the hCaR as judged by lack of cell surface expression of T706 and T806 mutants. T888 and T907 mutants with 25 and 40 residues, respectively, of carboxy-terminal tail showed identical expression and signal transduction as the wild type receptor. T865 and T874 mutants, with 2 and 11 amino acid long carboxy-terminal tails,respectively, showed a reduction in cell surface expression and no phosphoinositide response to extracellular calcium. However, reduced stimulation of phosphoinositol accumulation was far more dramatic than the corresponding reduction in receptor expression. We conclude that the membrane-proximal portion ofthe hCaR carboxy-terminal tail plays a key role in normal expression of the receptor and may include amino acid residues importemt for G protein (Gq/11) activation.

UR - http://www.scopus.com/inward/record.url?scp=33750174190&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33750174190&partnerID=8YFLogxK

M3 - Article

AN - SCOPUS:33750174190

VL - 11

JO - FASEB Journal

JF - FASEB Journal

SN - 0892-6638

IS - 9

ER -