Intermolecular interactions between dimeric calcium-sensing receptor monomers are important for its normal function

Abstract

We recently demonstrated that the G protein-coupled, extracellular calcium-sensing receptor (CaR) forms disulfide-linked dimers. The functional significance of dimerization of this receptor was suggested by our earlier observations that CaRs carrying certain point mutations exert dominant negative effects on the function of the coexpressed wild-type receptor both in vivo and when cotransfected in human embryonic kidney cells. In this study, we explored the functional consequences of CaR dimerization. Coexpression in human embryonic kidney cells of specific pairs of mutant CaRs, each with reduced or absent activity because of distinct loss-of-function mutations, results in the formation of heterodimers and partially reconstitutes extracellular calcium-dependent signaling. Moreover, our results suggest that the CaR has at least two functionally separable domains. However, the presence of an abnormal domain in each mutant monomer substantially impairs the function of the CaR heterodimer, resulting in the reconstituted CaRs having characteristics distinct from those of the wild-type CaR. Our study suggests that intermolecular interactions within the dimeric CaR are important for the receptor's function.

Figure 1

Cotransfection of inactive CaRs reconstitutes CaR-mediated, [Ca²⁺]_o-elicited [Ca²⁺]_i responses in HEK cells. Responses are normalized to the maximal cumulative [Ca²⁺]_i responses of cells transfected with wt alone for both A and B. (A) HEK cells were transfected with either wt or a mutant CaR, G143E, or E297K. (B) Cells were transfected with A877Stop or were cotransfected with A877Stop and the full-length wt (wt/A877Stop) or a mutant CaR, either G143E (G143E/A877Stop) or E297K (E297K/A877Stop). Points are mean values ± SEM) (n = 3–9).

Figure 2

Cotransfection of inactive CaRs reconstitutes CaR-mediated, [Ca²⁺]_o-elicited IP responses in HEK cells. Responses are normalized to the maximal IP responses of cells transfected with wt alone. (A) HEK cells were transfected with either wt or a mutant CaR, G143E, or E297K. (B) Cells were transfected with A877Stop and wt or were cotransfected with A877Stop and a mutant CaR, G143E (G143E/A877Stop) or E297K (E297K/A877Stop). Points are mean values ± SEM (n = 4).

Figure 3

(A) Detection of surface expression of dimeric mutant CaRs G143E and E297K. (B and C) Coimmunoprecipitation of A877Stop, a truncated CaR, and full-length mutant CaRs. HEK cells transfected with Flag-tagged CaRs or cotransfected with Flag-tagged and nontagged CaRs (as indicated in the figure) were biotinylated and lysed in the presence of 100 mM iodoacetamide. After immunoprecipitation with anti-Flag antibody, elution with SDS sample buffer without DTT (A and C) or with DTT (B) and SDS/PAGE (3–10%), CaR surface expression was detected with avidin.

Figure 4

Enhancement of the [Ca²⁺]_o-sensing capacity of the mutant CaR, R185Q, after its cotransfection with the truncated CaR, A877Stop. [Ca²⁺]_o-elicited [Ca²⁺]_i responses in HEK cells transfected with R185Q or cotransfected with R185Q and A877Stop were measured and normalized to the maximal cumulative [Ca²⁺]_i responses of cells transfected with wt alone. Points are mean values ± SEM (n = 4).

Figure 5

Effects of the point mutation R795W within i3 on the function of heterodimeric CaRs. (A) [Ca²⁺]_o-elicited [Ca²⁺]_i responses in HEK cells transfected with R795W or cotransfected with R795W and A877Stop were measured. (B) [Ca²⁺]_o-elicited [Ca²⁺]_i responses in HEK cells cotransfected with G143E and a CaR bearing a mutation within i3 and/or the tail, such as A877Stop, R795W, and A877Stop&R795W, were measured. All the measurements were normalized to the maximal cumulative [Ca²⁺]_i responses of cells transfected with wt alone. Points are mean values ± SEM (n = 4).

Figure 6

Coimmunoprecipitation of the mutant CaR, R795W, with the truncated CaR, A877Stop (A), and an inactive truncated CaR (A877Stop or R795W&A877Stop) with the inactive full-length mutant CaR, G143E (B). HEK cells transfected or cotransfected with Flag-tagged and nontagged CaRs (as indicated in the figure) were biotinylated before cell lysis in the presence of 100 mM iodoacetamide. After immunoprecipitation with anti-Flag antibody, elution with SDS sample buffer with DTT, and SDS/PAGE (3–10%), CaR surface expression was detected with avidin.