Identification of Amino Acid Residues That Direct Differential Ligand Selectivity of Mammalian and Nonmammalian V1a Type Receptors for Arginine Vasopressin and Vasotocin: INSIGHTS INTO MOLECULAR COEVOLUTION OF V1a TYPE RECEPTORS AND THEIR LIGANDS

Sujata Acharjee,Jean-Luc Do-Rego,Da Young Oh,Ryun Sup Ahn,Han Choe,Hubert Vaudry,Kyungjin Kim,Jae Young Seong,Hyuk Bang Kwon

doi:10.1074/jbc.m408909200

Abstract

Arginine vasotocin (VT) is the ortholog in all nonmammalian vertebrates of arginine vasopressin (AVP) in mammals. We have previously cloned an amphibian V1atype vasotocin receptor (VT1R) that exhibited higher sensitivity for VT than AVP, while the mammalian V1a type receptor (V1aR) responded better to AVP than VT. In the present study, we identified the amino acid residues that confer differential ligand selectivity for AVP and VT between rat V1aR and bullfrog VT1R (bfVT1R). A chimeric rat V1aR having transmembrane domain (TMD) VI to the carboxyl-terminal tail (C-tail) of bfVT1R showed a reverse ligand preference for AVP and VT, whereas a chimeric VT1R with TMD VI to the C-tail of rat V1aR showed a great increase in sensitivity for AVP. A single mutation (Ile(315(6.53)) to Thr) in TMD VI of V1aR increased the sensitivity for VT, while a single mutation (Phe(313(6.51)) to Tyr or Pro(334(7.33)) to Thr) reduced sensitivity toward AVP. Interestingly the triple mutation (Phe(313(6.51)) to Tyr, Ile(6.53) to Thr, and Pro(7.33) to Thr) of V1aR increased sensitivity to VT but greatly reduced sensitivity to AVP, behaving like bfVT1R. Further, like V1aR, a double mutant (Tyr(306(6.51)) to Phe and Thr(327(7.33)) to Pro) of bfVT1R showed an increased sensitivity to AVP. These results suggest that Phe/Tyr(6.51), Ile/Thr(6.53), and Pro/Thr(7.33) are responsible for the differential ligand selectivity between rat V1aR and bfVT1R. This information regarding the molecular interaction of VT/AVP with their receptors may have important implications for the development of novel AVP analogs.

Highlights

Arginine vasotocin (VT) is the ortholog in all nonmammalian vertebrates of arginine vasopressin (AVP) in mammals
These results suggest that Phe/Tyr6.51, Ile/Thr6.53, and Pro/Thr7.33 are responsible for the differential ligand selectivity between rat V1a type receptor (V1aR) and bullfrog VT1R (bfVT1R)
It is of interest to note that V1aR exhibits relatively high sensitivity to both AVP and VT, AVP is slightly more transmembrane domain; ICL, intracellular loop; ECL, extracellular loop; C-tail, carboxyl-terminal tail

Summary

EXPERIMENTAL PROCEDURES

Peptides—Synthetic peptides including AVP, oxytocin (OT), VT, mesotocin (MT), the V1 agonist [Phe2,Orn8]OT, the V2 agonist [deaminoCys1,Val4,D-Arg8]-AVP, the V1a antagonist [d(CH2)51,Tyr(Me)2]AVP, the V2 antagonist [d(CH2)51,D-Ile2,Ile4]AVP, and the OT antagonist [d(CH2)51,Tyr(Me)2,Thr4,Orn8,des-Gly-NH29]VT were purchased from Bachem (Bubendorf, Switzerland). The full-length bfVT1R cDNA was cloned into the pcDNA3 expression vector at the EcoRI and XhoI enzyme sites as mentioned earlier [25]. The rat V1aR cDNA was amplified through PCR from rat liver tissue and inserted at the EcoRI and XhoI sites of the pcDNA3. All the chimeric constructs were cloned into the pcDNA3 expression vector at the EcoRI and XhoI sites. For c-fos promoter-driven luciferase assay, cells were maintained in serum-free Dulbecco’s modified Eagle’s medium 16 –18 h before treatment with the ligand as described previously [32]. The plasmids containing the VT1R chimeric receptors were cotransfected with the c-fos-luc reporter vector into CV-1 cells along with ␤-galactosidase as an internal control.

RESULTS

Receptors and mutants

ND ND ND

DISCUSSION