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

Abstract

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.

Transmembrane domain IV of the Gallus gallus VT[sub 2] vasotocin receptor is essential for forming a heterodimer with the corticotrophin releasing hormone receptor

“…17 Moreover, it was demonstrated that human V1bR and CRHR1 can form constitutive homoand heterodimers. 26 An atomic-force microscopy analysis of native retinal disk membranes has revealed an organization of molecules of the prototypic GPCR rhodopsin in paracrystalline arrays.…”

“…17 The logical next step to extend this observation would be to find the dimerization interfaces between the two receptors. The three-dimensional architectures of VT2R and CRHR have not been solved, as GPCRs are inherently hard to crystallize due to their relatively insoluble, lipophilic TM regions.…”

“…17 Mutations of Ala 206 to Lys in the fluorescent part of pECFP-N1 and pEYFP-N1 expression vectors were made by using the Quick Change site-directed mutagenesis kit (Stratagene), a iCycler Thermal Cycler (BIO-RAD Laboratories, Inc.), and PfuTurbo DNA polymerase (Stratagene). The 50 μl reaction mixtures each contained approximately 40 ng plasmid template, 125 ng each oligonucleotide primer sense A206K top: 5′-CAG TCC AAG CTG AGC AAA GAC CCC AAC GAG AAG CGC GAT CAC-3′ and antisense A206K bottom: 5′-GTG ATC GCG CTT CTC GTT GGG GTC TTT GCT CAG CTT GGA CTG-3′ 19 (Integrated DNA Technologies), each deoxynucleoside triphosphate (Stratagene) in a final concentration of 0.05 mM, and 2.5 U PfuTurbo DNA polymerase.…”

“…17 Briefly, AVT and CRH were diluted into modified receptor buffer (10 mM KCl, 10 mM MgCl 2 , 2 mM EGTA, 20 mM Hepes, 120 mM NaCl, 1 mg/ml BSA, pH 7.4) as previously suggested. 20 HeLa cells transiently transfected with CRHR-, VT2R-, and VT2/β 2 AR-fusion protein constructs were incubated with 100 nM human CRH and 100 nM (Arg8)-vasotocin for 20 min at 37° C.…”

The development of endotoxin tolerance, and the role of hypothalamo-pituitary-adrenal function and glucocorticoids in Pekin ducks

“…In birds, the amount of CORT that can be elicited by various HPA stimulants provides a measure of HPA sensitivity (Kuenzel and Jurkevich, 2010;Mikhailova et al, 2007;Spelman et al, 1995). We used the protocol of Spelman et al (Spelman et al, 1995) to test for adrenal sensitivity with an intravenous (i.v.)…”