The carboxyl terminus of the human calcium receptor. Requirements for cell-surface expression and signal transduction.
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 approximately 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 approximately 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.
Ray
K
K
Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Fan
G F
GF
Goldsmith
P K
PK
Spiegel
A M
AM
D000581:Amidohydrolases / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D016296:Mutagenesis; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010716:Phosphatidylinositols / Q000378:metabolism; D010902:Pituitary Gland / Q000737:chemistry; D017433:Protein Structure, Secondary; D015398:Signal Transduction; D013499:Surface Properties; D014162:Transfection
D002135:Calcium-Binding Proteins; D010716:Phosphatidylinositols; D006596:Hexosaminidases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase