Charged residues at the intracellular boundary of transmembrane helices 2 and 3 independently affect constitutive activity of Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor

Because charged residues at the intracellular ends of transmembrane helix ( TMH) 2 and TMH3 of G protein-coupled receptors (GPCRs) affect signaling, we performed mutational analysis of these residues in the constitutively sign aling Kaposi's sarcoma-associated herpesvirus GPCR (KSNV-GPCR), KSHV-GPCR c ontains the amino acid sequence Val-Arg-Tyr rather than the Asp/Glu-Arg-Tyr ((D/ E)RY) moth at the intracellular end of TMH3, Mutation of Arg-143 to A la (R143A) or Gln (R143Q) abolished constitutive signaling whereas R143K ex hibited 50% of the basal activity of RSHV-GPCR, R143A was not stimulated by agonist, whereas R143Q was stimulated by growth-related oncogene-cu, and R 143K, similar to KSHV-GPCR, was stimulated further. These findings show tha t Arg-143 is critical for signal generation in KSHV-GPCR In other GPCRs, Ar g in this position may act as a signaling switch by movement of its sidecha in from a hydrophilic pocket in the TMH bundle to a position outside the bu ndle. In rhodopsin, the Arg of Glu-Arg-Tyr interacts with the adjacent Asp to constrain Arg outside the TMH bundle. V142D was 70% more active than KSH V-GPCR, suggesting that an Arg residue, which is constrained outside the bu ndle by interacting with Asp-142, leads to a receptor that signals more act ively, Because the usually conserved Asp in the middle of TMH2 is not prese nt in KSHV-GPCR, we tested whether Asp-83 at the intracellular end of TMH2 was involved in signaling. D83N and D83A were 110 and 190% more active than KSHV-GPCR, respectively. The double mutant D83A/V142D was 510% more active than KSHV-GPCR That is, cosubstitutions of Asp-83 by Ala and Val-142 by As p act synergistically to increase basal signaling. A model of RSHV-GPCR pre dicts that Arg-143 interacts with residues in the TMH bundle and that the s idechain of Asp-83 does not interact with Arg-143, These data are consisten t with the hypothesis that Arg-143 and Asp-83 independently affect the sign aling activity of KSHV-GPCR.