Dimerization of the Extracellular Calcium-sensing Receptor (CaR) on the Cell Surface of CaR-transfected HEK293 Cells

Abstract

The extracellular calcium (Ca 2؉ o )-sensing receptor (CaR) is a G protein-coupled receptor that plays important roles in calcium homeostasis. In this study, we employed epitope tagging, cell-surface biotinylation, and immunoprecipitation techniques to demonstrate that the CaR is expressed mostly in the form of a dimer on the surface of transfected human embryonic kidney (HEK293) cells. Western analysis of cell-surface proteins under nonreducing conditions showed that the CaR exists in several forms with molecular masses greater than 200 kDa. Most of these high molecular mass forms of the receptor could be converted to a single monomeric species at 160 kDa under reducing conditions. This result suggests that the CaR forms dimers or even higher oligomers on the cell surface through intermolecular disulfide bonds that are sensitive to reducing agents. Consistent with this hypothesis, use of a cell-surface crosslinking agent substantially increases the proportion of the putative dimeric CaR at 280 kDa relative to the monomeric form of the receptor at 160 kDa under reducing conditions. Dimerization of the CaR in intact cells was further demonstrated when we co-transfected and co-immunoprecipitated the wild type, full-length receptor and a truncated form of the CaR lacking its cytoplasmic tail. Taken together, we conclude from these results that the functional CaR resides on the cell surface of transfected HEK293 cells in the form of a dimer.

Modeling and Mutagenesis of the Binding Site of Calhex 231, a Novel Negative Allosteric Modulator of the Extracellular Ca2+-sensing Receptor

“…Our results are in agreement with previous carbohydrate analysis of the human receptor expressed in HEK293 cells showing that the polypeptides of higher molecular mass correspond to N-linked glycosylated receptors expressed at the cell surface, whereas the polypeptides of lower molecular mass represent intracellular mannose-modified receptors (40). A polypeptide complex migrating above 200 kDa was also identified and might correspond to intermolecular linked dimers as previously observed (40). Importantly, the expression pattern of the different mutant receptors was comparable to that of the WT receptor as accessed by immunoblotting (Fig.…”

Hypercalcaemic and hypocalcaemic conditions due to calcium-sensing receptor mutations

“…35 The functional cell surface form of the CaSR is a dimer, and the two monomers within the dimeric CaSR are linked by disulphide bonds involving cysteine residues 129 and 131 within each monomer. 36 The ECD of each CaSR monomer probably contains more than one binding site for Ca 2+ o because the Hill coefficient for the activation of the receptor by Ca 2+ o is 3-4, consistent with the presence of positive cooperativity amongst at least this number of binding sites within the dimeric CaSR. 37, 38 The TMD also appears to be involved in Ca 2+ o sensing, since a mutant CaSR lacking the ECD also responds to Ca 2+ o and other polyvalent cations.…”

Ligand-independent CXCR2 Dimerization

“…Receptor oligomerization has functional implications in terms of cell surface expression, ligand binding, signaling, and receptor trafficking (1). Many different GPCRs have been proved to undergo homoor heterodimer formation; these include the receptors for dopamine which can form both homo-(2-4) and heterodimers with somatostatin receptors (5), angiotensin A1 and A2 (6), adrenergic (7,8), GABA B (9 -11), ␦-and -opioid (12)(13)(14)(15), rhodopsin (16), mGlu (17), vasopressin V2 (18), vasopressin/ossitocin (19), muscarinic (20), glucagon (21), Ca 2ϩ (22) sphingosine 1-phosphate (23), and the chemokine CXCR4, CCR2, and CCR5 receptors (24 -28). The molecular mechanism of receptor oligomerization varies among different receptors and involves transmembrane (TM) domains as well as extramembrane regions.…”

“…The molecular mechanism of receptor oligomerization varies among different receptors and involves transmembrane (TM) domains as well as extramembrane regions. Cysteine residues in the fourth TM segment are responsible for D2 dopamine receptor dimerization (4), and disulfide bonds are also involved in the oligomerization of Ca 2ϩ (22), -opioid (13), sphingosine 1-phosphate (23), and V2 vasopressin receptors (18); sequences in the N-terminal extracellular domains are involved in mGluR (17) and CCR5 (24 -27) homodimer formation; the C-terminal region is essential for ␦-opioid receptor dimerization (12). The seventh TM domain controls noncovalent hydrophobic interactions for adrenergic receptors, which also require receptor glycosylation (7,8).…”