Type 2B corticotrophin releasing factor receptor forms a heteromeric complex with dopamine D1 receptor in living cells

Abstract

Corticotrophin releasing factor (CRF) and its related peptides differentially bind to CRF receptors to modulate stress-related behaviors. CRF receptors comprise two G-protein coupled receptors (GPCR), type-1 CRF receptors (CRF1), and type-2 CRF receptors (CRF2). CRF2 encompasses three spliced variants in humans, alpha (CRF2a), beta (CRF2b), and gamma (CRF2g), which differ in their N-terminal extracellular domains and expression patterns. Previously, we showed that CRF2a form a heteromeric protein complex with dopamine D1 receptors (D1R), leading to changes in the signaling of D1R. Based on the high sequence identity between CRF2a and CRF2b, we hypothesized that CRF2b also heteromerize with D1R. To test the hypothesis, we compared the expression and localization of both CRF2 isoforms and whether CRF2b form stable protein complexes with D1R in HEK293 and ATR75 cell lines. We observed that the immunoreactivity for CRF2b was similar to that of CRF2a in the endoplasmic compartment but significantly higher in the Golgi compartment. Immunoprecipitation analysis showed that CRF2b forms a heteromeric protein complex with D1R. Furthermore, the protein complex formed by CRF2b and D1R was stable enough to change the sub-cellular localization of CRF2b when it was co-expressed with a construct of D1R bearing a nuclear localization signal. Immunofluorescence in A7R5 cells, which endogenously express CRF2b and D1R, shows significant colocalization of CRF2b with D1R. In conclusion, our results show that CRF2b forms a stable heteromeric protein complex with D1R, a potential new therapeutic target in tissues where both receptors are co-expressed, such as the septum in the brain, and heart, kidney, and skeletal muscle in the periphery.