Dimerization between vasopressin V1b and corticotropin releasing hormone type 1 receptors

Abstract

1. Increasing evidence indicates that guanyl protein coupled receptors (GPCRs), including members of the vasopressin (VP) receptor family can act as homo- and heterodimers. Regulated expression and interaction of pituitary VP V1b receptor (V1bR) and corticotropin releasing hormone receptor type 1 (CRHR1) are critical for hypothalamic pituitary adrenal (HPA) axis adaptation, but it is unknown whether this involves physical interaction between these receptors.2. Bioluminescence resonance energy transfer (BRET) experiments using V1bR and CRHR1 fused to either Renilla luciferase (Rluc) or yellow fluorescent protein (YFP) at the N-terminus, but not the carboxyl-terminus, revealed specific interaction (BRET(50) = 0.39 +/- 0.08, V1bR) that was inhibited by untagged V1b or CRHR1 receptors, suggesting homo- and heterodimerization. The BRET data were confirmed by coimmunoprecipitation experiments using fully bioactive receptors tagged at the aminoterminus with c-myc and Flag epitopes, demonstrating specific homodimerization of the V1b receptor and heterodimerization of the V1b receptor with CRHR1 receptors.3. Heterodimerization between V1bR and CRHR1 is not ligand dependent since stimulation with CRH and AVP had no effect on coimmunoprecipitation. In membranes obtained from cells cotransfected with CRHR1 and V1bR, incubation with the heterologous nonpeptide antagonist did not alter the binding affinity or capacity of the receptor.4. The data demonstrate that V1bR and CRHR1 can form constitutive homo- and heterodimers and suggests that the heterodimerization does not influence the binding properties of these receptors.

Fig. 1.

Biological activity of tagged V1b and CRHR1 receptors. (A) Representative confocal images of CHO cells transfected with V1b receptor tagged at either the C-terminus or N-terminus with yellow fluorescent protein (V1bCTYFP and V1bNTYFP, respectively) or CRHR1 tagged at the C-terminus with YFP (CRHRCTYFP). (B) Representative AVP (10 nM) induced increases in intracellular calcium in CHO cells transfected with N-terminus-tagged V1b receptors with myc (V1bmyc) or Flag (V1bFlag) and C-terminus Renilla luciferase (V1bCTRluc) or YFP (V1bCTYFP), compared to wild-type receptor (V1bWT). Data points are the mean of duplicate determinations in one of three experiments. (C) CRH dose response for beta-galactosidase activity in the reporter cell line, LVIP2.0Zc, transfected with Flag- (CRHRFlag) or YFP- (CRHRCTYFP) tagged CRHR1 receptors, compared to wild-type CRHR (CRHRWT) receptor. Data points are the mean and SE of beta galactosidase activity values (absorbance) in three separate experiments.

Fig. 2.

V1b receptor homodimerization assessed by bioluminescence resonance energy transfer (BRET). CHO cells were transfected with 100 ng of V1b tagged with renilla luciferase at the N- or C-terminus (V1bNTRluc or V1bCTRluc) with or without 100 ng V1b tagged with YFP at the N- or C-terminus (V1bNTYFP or V1bCTYFP). Rluc (480 nm emission) and YFP (530 nm emission) signals were measured before and after injection of coelenterazine h, using a multiplate reader. BRET was determined by the equation: BRET ratio = (530 nm_exp/480 nm_exp) − (530 nm_background/480 nm_background), where exp is cells transfected with both Rluc- and YFP-tagged receptors and background is cells transfected with Rluc-tagged receptor only. Bars represent the mean and SE of data obtained in three experiments. ^* P < 0.05 compared with other groups.

Fig. 3.

Specificity of V1b receptor homodimerization and V1b/CRHR1 heterodimerization. (A) BRET saturation curve performed in CHO cells transfected with 100 ng of V1bNTRluc with or without increasing amounts (10–1000 ng) of V1bNTYFP. (B) BRET competition curve. CHO cells were transfected with V1bNTRluc and V1bNTYFP (100 ng each) plus increasing amounts of either the V1bmyc (N-terminus tag) or wild-type CRHR1 (CRHR1WT) or 100 and 200 ng of wild-type V1b receptor (V1bWT). BRET ratios were measured as described in Methods and in the legend to Fig. 2. Data are represented as the mean ± SEM of data obtained in three experiments.

Fig. 4.

Representative blot from coimmunoprecipitation of V1b receptor homodimers. Total protein lysates (500 μg) from CHO cells, untransfected, or cotransfected with N-terminus-tagged V1bFlag and V1bmyc (V1bmyc + V1bFlag), or pooled lysates from cells transfected with the individual constructs (V1bmyc & V1bFlag mix) were immunoprecipitated with Flag antibody (IP: Flag) and immunoblotted with anti-myc (IB: myc) to determine homodimerization or with anti-Flag (IB: Flag) to confirm immunoprecipitation. Coimmunoprecipitation was confirmed in three separate transfections. Specific V1b receptor bands are shown by the open arrow.

Fig. 5.

Effect of the ligand on V1b homodimerization. CHO cells were transfected with 2 μg of N-terminus-tagged V1bmyc + 2μg V1bFlag (homodimer), or transfected with either 2 μg V1bmyc + 2 μg empty vector, or 2 μg V1bFlag + 2 μg empty vector and pooled after lysis (Neg. Control). Twenty-four hours posttransfection, cells were incubated with either DMEM/0.1%BSA (Vehicle) or 10⁻⁷ M AVP (AVP), for 10 min at 37°C, prior lysis. Immunoprecipitation with anti-Flag (IP: Flag) and immunoblot with anti-myc (IB: myc, to detect homodimers) or anti-Flag (IB: Flag, to confirm immunoprecipitation) was carried out as described in Methods. The specific V1b receptor bands are shown by the open arrows.

Fig. 6.

Representative Western blot of tagged CRHR1 after immunoprecipitation of homodimers. Total cell protein aliquots (500 μg) from CHO cells, either from cells cotransfected with 2 μg of each CRHRFlag and CRHRCTYFP (CRHRFlag + CRHRCTYFP), or a 1:1 mix of protein from cells transfected with CRHRFlag + empty vector, or CRHRCTYFP + empty vector (CRHRFlag & CRHRCTYFP mix), or untransfected CHO cells, were immunoprecipitated with GFP antibody (IP: GFP). Immunoblots were performed with anti-Flag (IB: Flag) to determine homodimerization, and with anti-GFP (which equally cross react with YFP) (IB: GFP) to confirm immunoprecipitation. Coimmunoprecipitation was confirmed with two separate transfections. Specific bands for CRHRFlag (MW ∼45) or CRHRCTYFP bands are shown by the open arrows.

Fig. 7.

Representative blots of V1b/CRHR1 coimmunoprecipitation. (A) Aliquots containing 500 μg of lysates from CHO cells cotransfected with 2 μg V1bmyc + 2 μg CRHRWT (V1bmyc + CRHR WT), or a 1:1 mixture of proteins from cells individually transfected with V1bmyc + 2 μg empty vector or 2 μg CRHR1WT + 2 μg empty vector (V1bmyc and CRHR WT mix), or untransfected cells were immunoprecipitated with anti-CRHR antibody (IP: CRHR). Immunoblot with anti-myc (IB: myc) was performed to determine heterodimerization, and with anti-CRHR (IB: CRHR) to confirm immunoprecipitation. Coimmunoprecipitation was confirmed in three separate transfections. (B) V1b/CRHR1 heterodimerization specificity was determined by competition with increasing amounts (0–2 μg) of wild-type V1b receptor in cells cotransfected with 1 μg each of V1bmyc and CRHR1WT. A 1:1 mixture of proteins from cells transfected individually with either V1bmyc or CRHR1WT, and untransfected CH) cells were used as negative controls. Coimmunoprecipitation was confirmed in two separate transfections performed in duplicate.

Fig. 8.

Effect of heterologous ligand on the binding properties of CRHR1/V1bR heterodimers. (A) [³H]AVP binding saturation curves in the presence and in the absence of 10 μM of CRH or the nonpeptide CRHR1 antagonist, DMP-696. Binding curves were performed by incubation of 10 μg membrane protein from CHO cells cotransfected with V1b and CRH receptor plasmids (2 ug DNA of each in 75 cm²) with 0.1 to 6 nM of [³H]AVP, in the absence or presence or the V1b receptor antagonist, 1 μM SSR149415 (nonspecific), at room temperature for 60 min. (B) Binding saturation curves for [¹²⁵I]Sauvagine, in the presence and in the absence of AVP, or 1 μM of the nonpeptide V1b receptor antagonist, SSR149415. Binding curves were performed by incubating 10 μg of membrane protein from cotransfected CHO cells with [¹²⁵I]sauvagine and increasing concentrations of unlabeled sauvagine, for 1 h at room temperature (nonspecific binding was measured in the presence of 10 μM DMP-696. Data points are the mean of duplicate incubations in a representative experiment.