The Role of Cysteines and Charged Amino Acids in Extracellular Loops of the Human Ca²⁺Receptor in Cell Surface Expression and Receptor Activation Processes

Abstract

The Ca(2+) receptor is a plasma-membrane bound G protein-coupled receptor stimulated by extracellular calcium [Ca(2+)](o) and other di- and poly-cations. We investigated the role in receptor activation of all the charged amino acid residues and cysteines in the three extracellular loops (EL1, 2, and 3) of the human Ca(2+) receptor by alanine-scanning mutagenesis. The mutant receptors were transiently expressed in HEK-293 cells, and cell surface expression patterns were analyzed by endoglycosidase-H digestion, immunoblotting, intact cell ELISA, and hydrolysis of phosphoinositides (PI) induced by [Ca(2+)](o.) The mutation of Cys677 and Cys765 located in EL1 and EL2, respectively, ablated PI hydrolysis completely, showed less than 5% cell surface expression of the wild-type receptor, and were not properly glycosylated. Replacement of the charged residues by using a single mutation or multiple alanine mutations in EL1, 2, and 3 produced only minor changes in receptor activation, except for Glu767 and Lys831. The E767A and K831A mutations in EL2 and EL3, respectively, showed gain-of-function by significantly enhancing apparent [Ca(2+)](o) affinity. E767A and K831A exhibited EC(50) values of 2.1 and 2.8 mm, respectively, for [Ca(2+)](o)-stimulated PI hydrolysis as opposed to EC(50) value of 4.2 mm for the wild-type receptor. Like E767A, substitutions of Glu767 with Gln and Lys was similarly activating, whereas Asp substitution displayed wild-type [Ca(2+)](o) sensitivity. Substitution of Lys831 with Glu but not with Gln showed similar activating effect as Ala replacement. A double-mutant E767K/K831E in which charged residues were switched positions showed impaired cell surface expression and failed to respond to [Ca(2+)](o.) Taken together, these results suggest that in ELs, two cysteines form critical disulfide links, and the side chains of Glu767 and Lys831 are probably involved in ionic interactions with other prospective oppositely charged residues. Some of these interactions could be important for receptor folding and also may contribute to keep the Ca(2+) receptor transmembrane helix bundle in an inactive conformation.

The endoplasmic reticulum‐associated protein, OS‐9, behaves as a lectin in targeting the immature calcium‐sensing receptor

“…We next examined the ability of OS‐9‐1 to bind to a previously described CaSR mutant (C677A) that remains trapped in the ER due to a predicted misfolding defect (Ray et al, ). Indeed, in Western blots, this mutant reveals only the immature mannose form of the receptor—the mature glycosylated receptor is not produced indicating that it is neither processed by the Golgi nor trafficked to the cell surface (Ray et al, ). We hypothesized that this CaSR mutant might be preferentially targeted by OS‐9.…”

“…The CaSR‐FLAG‐del(895–1075) is expressed in mature form, can reach the cell surface and is functional (Lienhardt et al, ), so it is probable that it has a similar ability to fold as wild‐type receptor. On the other hand, the mutant CaSR‐C677A, ER‐trapped mutant has been predicted to fold improperly (Ray et al, ) suggesting that it would be subject to enhanced trimming of N ‐glycans to favor OS‐9 binding. Surprisingly, however, we did not observe preferential binding of CaSR‐C677A to OS‐9.…”

“…Following sequence validation, the PCR product was subcloned into pcDNA3.1‐CaSR‐FLAG via the unique restriction sites, BsrGI and XbaI. The C677A mutation (Ray et al, ) was generated by site‐directed mutagenesis using pcDNA3.1‐exFLAG‐CaSR as template. Following sequence validation the mutation was cassetted back into pcDNA3.1‐CaSR‐FLAG using the unique CaSR enzymes BsrGI and BamHI to generate pcDNA3.1‐CaSR(C677A)‐FLAG (note that the unique Bam HI site in the poly‐cloning region of pcDNA3.1 was removed in the construction of pcDNA3.1‐CaSR‐FLAG).…”

“…The CaSR is composed of four major domains, a large amino terminal extracellular, Venus‐flytrap domain for the binding of nutrients, a cysteine‐rich domain, a characteristic hepta‐helical membrane domain, and a large intracellular carboxy‐terminal tail (Hu & Spiegel, ). Proper glycosylation and dimerization of the receptor and disulphide bond formation between cysteines in the extracellular loops of the CaSR are required to generate a fully functional cell surface receptor (Bai, Trivedi, & Brown, ; Ray, Clapp, Goldsmith, & Spiegel, ; Ray, Ghosh, & Northup, ). Dimerization occurs in the endoplasmic reticulum (ER) (Pidasheva et al, ) and is in part mediated by intermolecular, covalent bonds formed between cysteine residues 129 and 131 and in part by noncovalent, interactions between leucine residues 112 and 156 (Jiang, Minet, Zhang, Silver, & Bai, ; Ray et al, ).…”

Calindol, a Positive Allosteric Modulator of the Human Ca2+ Receptor, Activates an Extracellular Ligand-binding Domain-deleted Rhodopsin-like Seven-transmembrane Structure in the Absence of Ca2+

“…It would appear that in the intact hCaR structure the presence of the ECD prevents Calindol from activating the 7TMD. We previously suggested a strong allosteric coupling between the ECD and 7TMD, probably conveyed through interactions with the exo-loops, as a basis for Ca 2ϩ binding in the ECD allosterically activating the 7TMD for G-protein coupling (31 (14). However, we and others (16,21,32) have reported that, whereas Ca 2ϩ alone produces very little or no activation of the ECD-truncated T903-Rhoc construct, in the presence of NPS R-568 and as shown in this study, in the presence of Calindol, Ca 2ϩ dramatically stimulates the 7TMD hCaR construct.…”