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 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.
Highlights
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 ϳ25% reduction in cell-surface expression
The wild-type human CaR (hCaR) cDNA and each of the mutant constructs were transiently transfected into HEK-293 cells, and expression was assessed by immunoblotting using a monoclonal antibody (ADD) raised against a peptide epitope from the extracellular domain of the receptor that was not disrupted by the truncations (Fig. 2)
This study provides new information on the role of membrane-spanning domains and the carboxyl-terminal tail in hCaR processing, cell-surface expression, and signal transduction
Summary
CaR, calcium receptor; hCaR, human calcium receptor; GPCR, G-protein-coupled receptor; Ca2ϩo, extracellular calcium ion; PI, phosphoinositide; ECD, extracellular domain; FBHH, familial benign hypocalciuric hypercalcemia; PCR, polymerase chain reaction; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline; PNGase F, peptide N-glycosidase F; Endo-H, endo--N-acetylglucosaminidase H; PIPES, 1,4-piperazinediethanesulfonic acid. Given the unique sequence and predicted topology of the hCaR and the dearth of naturally occurring mutations involving the transmembrane domains and the carboxyl terminus of the receptor, we sought to define the importance of these regions in receptor biosynthesis, cell-surface expression, and signal transduction. For this purpose, we created a series of mutants that truncate or alter the receptor sequence within the second and third intracellular loops and within the carboxylterminal tail. Our results indicate that all seven transmembrane helices may be necessary for proper folding, processing, and cell-surface expression of the hCaR and define a membrane-proximal portion of the carboxyl-terminal tail that contains residues critical for cell-surface expression and signal transduction
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
