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Heterooligomerization between Vasotocin and Corticotropin-Releasing Hormone (CRH) Receptors Augments CRH-Stimulated 3',5'-Cyclic Adenosine Monophosphate Production

Departments of Physiology and Biophysics (M.V.M., L.E.C.), and Pharmacology and Toxicology (P.R.M.), University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; Department of Poultry Science (A.J., W.J.K., F.M.), University of Arkansas, Fayetteville, Arkansas 72701; and W.M. Keck Center for Cellular Imaging (A.P., Y.C.), University of Virginia, Charlottesville, Virginia 22904

Address all correspondence and requests for reprints to: Marina Mikhailova, Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, 4301 West Markham Street, Slot 750, Little Rock, Arkansas 72205. E-mail: MikhailovaMarinaV{at}uams.edu.

In birds, ACTH release from the anterior pituitary gland during stress is controlled by CRH and arginine vasotocin (AVT). Using 5-wk-old male chicks, simultaneous iv injections of CRH and AVT were found to result in a greater than additive increase in plasma corticosterone levels compared with that obtained with individual administration of either peptide hormone. In order to investigate molecular mechanisms underlying this observation, the chicken CRH receptor (CRHR) and vasotocin VT2 receptor (VT2R) were fused to cyan and yellow fluorescent proteins and expressed in HeLa cells. The resulting CRHR and VT2R fusion proteins were expressed appropriately in the plasma membrane and were found to couple to downstream signal transduction pathways. Quantitative fluorescence resonance energy transfer (FRET) analysis was used to determine whether the CRHR and VT2R formed heterodimers. In the absence of CRH and AVT, the FRET efficiency was 15–18%, and the distance between receptors was 5–6 nm. Treatment of the cells that expressed both cyan fluorescent protein-CRHR and yellow fluorescent protein-VT2R with CRH or AVT alone did not lead to a significant change in the FRET efficiency. However, simultaneous addition of these hormones increased the efficiency of the FRET signal and decreased the distance between the two receptors. In HeLa cells expressing both CRHR and VT2R, treatment with CRH and AVT resulted in a significant increase in cAMP production over that with CRH alone, indicating that heterodimer formation may enhance the ability of the CRHR to activate downstream signal transduction.