The carboxyl terminus of the human calcium receptor. Requirements for cell-surface expression and signal transduction

Kausik Ray, Gao Feng Fan, Paul K. Goldsmith, Allen M. Spiegel

Research output: Contribution to journalArticle

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Abstract

The G-protein-coupled calcium receptor plays a key role in extracellular calcium homeostasis. To examine the role of the membrane-spanning domains and the ~200-residue cytoplasmic carboxyl terminus of the calcium receptor in cell-surface expression and signal transduction, we transfected HEK-293 cells with a series of truncation and carboxyl-terminal missense mutants and analyzed expression by immunoblotting, glycosidase digestion, intact cell immunoassay, and extracellular calcium-stimulated phosphoinositide hydrolysis assay. Two truncation mutants terminating at residues 706 and 802 within the second and third intracellular loops, respectively, were not properly glycosylated, failed to reach the cell-surface, and showed no calcium response, indicating that mutant receptors with the full extracellular domain but only three or five transmembrane domains are improperly folded and/or processed. Truncation mutants terminating at residues 888 and 903 within the carboxyl terminus were equivalent to the wild type in all assays, whereas mutants truncated at residues 865 and 874 showed no response to calcium, despite only ~25% reduction in cell-surface expression. Mutants with a full- length carboxyl terminus but with residues between positions 874 and 888 replaced with alanines showed either no (Ala875, Ala876, and Ala879) or significantly reduced (Ala881-Ala883) calcium response at levels of cell-surface expression equivalent to those of the wild-type receptor. These results indicate that deletion of the majority of the carboxyl terminus is compatible with normal processing, cell-surface expression, and signal transduction of the receptor. The truncation and alanine substitution mutants identify a small region between residues 874 and 888 critical for normal signal transduction by the receptor.

Original languageEnglish (US)
Pages (from-to)31355-31361
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number50
DOIs
StatePublished - Dec 12 1997
Externally publishedYes

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Calcium-Sensing Receptors
Signal transduction
Signal Transduction
Calcium
Alanine
Assays
Glycoside Hydrolases
Phosphatidylinositols
GTP-Binding Proteins
HEK293 Cells
G-Protein-Coupled Receptors
Hydrolysis
Immunoassay
Immunoblotting
Substitution reactions
Digestion
Membranes
Homeostasis
Processing

ASJC Scopus subject areas

  • Biochemistry

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The carboxyl terminus of the human calcium receptor. Requirements for cell-surface expression and signal transduction. / Ray, Kausik; Fan, Gao Feng; Goldsmith, Paul K.; Spiegel, Allen M.

In: Journal of Biological Chemistry, Vol. 272, No. 50, 12.12.1997, p. 31355-31361.

Research output: Contribution to journalArticle

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