Dimerization Between Vasopressin V1b and Corticotropin Releasing Hormone Type 1 Receptors
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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 (BRET50 = 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.
KEY WORDSvasopressin V1b receptor corticotropin releasing hormone type 1 receptor dimerization BRET immunoprecipitation receptor binding
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