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 signaling Kaposi's sarcoma-associated herpesvirus GPCR (KSHV-GPCR). KSHV-GPCR contains the amino acid sequence Val-Arg-Tyr rather than the Asp/Glu-Arg-Tyr ((D/E)RY) motif at the intracellular end of TMH3. Mutation of Arg-143 to Ala (R143A) or Gln (R143Q) abolished constitutive signaling whereas R143K exhibited 50% of the basal activity of KSHV-GPCR. R143A was not stimulated by agonist, whereas R143Q was stimulated by growth-related oncogene-alpha, and R143K, similar to KSHV-GPCR, was stimulated further. These findings show that Arg-143 is critical for signal generation in KSHV-GPCR. In other GPCRs, Arg in this position may act as a signaling switch by movement of its sidechain from a hydrophilic pocket in the TMH bundle to a position outside the bundle. 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 KSHV-GPCR, suggesting that an Arg residue, which is constrained outside the bundle by interacting with Asp-142, leads to a receptor that signals more actively. Because the usually conserved Asp in the middle of TMH2 is not present 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 Asp act synergistically to increase basal signaling. A model of KSHV-GPCR predicts that Arg-143 interacts with residues in the TMH bundle and that the sidechain of Asp-83 does not interact with Arg-143. These data are consistent with the hypothesis that Arg-143 and Asp-83 independently affect the signaling activity of KSHV-GPCR.
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Division of Molecular Medicine, Department of Medicine, Weill Medical College and Graduate School of Medical Sciences of Cornell University, New York, New York 10021, USA.
Ganeshalingam
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N
Rosenhouse-Dantsker
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A
Osman
R
R
Gershengorn
M C
MC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D054377:Chemokine CXCL12; D018925:Chemokines / Q000494:pharmacology; D019742:Chemokines, CC / Q000494:pharmacology; D019743:Chemokines, CXC / Q000494:pharmacology; D017931:DNA Primers; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D019707:Receptors, Chemokine / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology
C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D018925:Chemokines; D019742:Chemokines, CC; D019743:Chemokines, CXC; D017931:DNA Primers; C115448:G protein-coupled receptor, Human herpesvirus 8; D019707:Receptors, Chemokine; D011994:Recombinant Proteins; D014764:Viral Proteins; C108059:vMIP-II