Species sequence differences determine the interaction of GnRH receptor with the cellular quality control system

Abstract

Plasma membrane expression (PME) of the human GnRHR (hGnRHR) is regulated by a primate-specific Lys(191) which destabilizes a Cys(14)-Cys(200) bridge required by the cellular quality control system (QCS). A 4-amino, non-contiguous "motif" (Leu(112), Gln(208), Leu(300), Asp(302)) is required for this effect. The hGnRHR sequence, with or without Lys(191), decreases PME and inositol phosphate (IP) production when co-expressed with calnexin, a QCS chaperone. WT rat GnRHR, decreases PME and IP production, when co-expressed with calnexin, but to a lesser degree than hGnRH. When the human sequence contains the rat motif, IP production is closer to that of rat GnRHR. When Lys(191) is deleted from hGnRHR and co-expressed with calnexin, IP production is similar to the rat sequence. When rat GnRHR containing Lys(191) and the human motif is co-expressed with calnexin, IP production is similar to cells expressing the hGnRHR. The motif sequence appears to be a determinant of calnexin recognition.

Keywords: Calnexin; ER protein retention; GPCR; GnRH receptor; Protein trafficking.

Fig. 1. Dose response curves of IP production by WT and mutant GnRH receptors in response to Buserelin

IP production was assessed in response to the indicated doses of Buserelin. A) COS-7 cells were transiently transfected with 25 ng of human WT GnRH receptor or mutant receptor plus 75 ng of empty vector (closed symbols) or human calnexin (open symbols). B) COS-7 cells were transiently transfected with 25 ng of rat WT GnRH receptor or mutant receptor plus 75 ng of empty vector (closed symbols) or rat calnexin (open symbols) as described in Materials and Methods. Means ± S.E.M.s are shown for three independent experiments, each performed in replicates of four.

Fig. 1. Dose response curves of IP production by WT and mutant GnRH receptors in response to Buserelin

IP production was assessed in response to the indicated doses of Buserelin. A) COS-7 cells were transiently transfected with 25 ng of human WT GnRH receptor or mutant receptor plus 75 ng of empty vector (closed symbols) or human calnexin (open symbols). B) COS-7 cells were transiently transfected with 25 ng of rat WT GnRH receptor or mutant receptor plus 75 ng of empty vector (closed symbols) or rat calnexin (open symbols) as described in Materials and Methods. Means ± S.E.M.s are shown for three independent experiments, each performed in replicates of four.

Fig. 2. Dose response curves of IP production by human WT and mutant GnRH receptors in response to Buserelin

IP production was assessed in response to the indicated doses of Buserelin. COS-7 cells were transiently transfected with 25 ng of WT or mutant receptors plus 75 ng of empty vector (closed symbols) or human calnexin (open symbols) as described in Materials and Methods. Means ± S.E.M.s are shown for three independent experiments, each performed in replicates of four.

Fig. 3. Dose response curves of IP production by WT and mutant GnRH receptors in response to Buserelin

IP production was assessed in response to the indicated doses of Buserelin. COS-7 cells were transiently transfected with 25 ng of WT or mutant plus 75 ng of empty vector (closed symbols) or human and rat calnexin (open symbols) as described in Materials and Methods. Panel A) hGnRHR, B) rat GnRHR and C) mouse GnRHR. Means ± S.E.M.s are shown for three independent experiments, each performed in replicates of four.

Fig. 3. Dose response curves of IP production by WT and mutant GnRH receptors in response to Buserelin

IP production was assessed in response to the indicated doses of Buserelin. COS-7 cells were transiently transfected with 25 ng of WT or mutant plus 75 ng of empty vector (closed symbols) or human and rat calnexin (open symbols) as described in Materials and Methods. Panel A) hGnRHR, B) rat GnRHR and C) mouse GnRHR. Means ± S.E.M.s are shown for three independent experiments, each performed in replicates of four.

Fig. 3. Dose response curves of IP production by WT and mutant GnRH receptors in response to Buserelin

IP production was assessed in response to the indicated doses of Buserelin. COS-7 cells were transiently transfected with 25 ng of WT or mutant plus 75 ng of empty vector (closed symbols) or human and rat calnexin (open symbols) as described in Materials and Methods. Panel A) hGnRHR, B) rat GnRHR and C) mouse GnRHR. Means ± S.E.M.s are shown for three independent experiments, each performed in replicates of four.

Fig. 4. Binding analysis using ¹²⁵I-Buserelin in COS-7 cells transfected with human and rat GnRHR and calnexin

The cells were transfected with 25 ng of human and rat WT or mutant receptors plus 75 ng of empty vector (solid bar) or human and rat calnexin (gray bar). Means ± S.E.M.s are shown for three independent experiments, each performed in replicates of four. *p < 0.05 vs same groups with calnexin and all groups no calnexin.

Fig. 5. Dose response curves of IP production by WT hGnRHR with phosphorylation mutant calnexin in response to Buserelin

IP production was assessed in response to the indicated doses of Buserelin. COS-7 cells were transiently transfected with 25 ng of WT hGnRHR plus 75 ng of empty vector (closed symbols) human calnexin mutant (open symbols) as described in Materials and Methods. Means ± S.E.M.s are shown for three independent experiments, each performed in replicates of four.