Elucidation of signaling properties of vasopressin receptor-related receptor 1 by using the chimeric receptor approach

Proc Natl Acad Sci U S A. 2004 Feb 10;101(6):1508-13. doi: 10.1073/pnas.0308250100. Epub 2004 Feb 2.

Abstract

The identification of endogenous or surrogate ligands for orphan G protein-coupled receptors (GPCRs) represents one of the most important tasks in GPCR biology and pharmacology. The challenge lies in choosing an appropriate assay in the absence of ways to activate the receptor of interest. We investigated the signaling pathway for an orphan GPCR referred to here as vasopressin receptor-related receptor 1 (VRR1) by generating a chimeric receptor, V1a/VRR1. The engineered construct contained vasopressin V1a receptor with all three intracellular loops and C terminus replaced by those of VRR1. The chimera behaved like a typical GPCR when transiently and stably expressed in mammalian cell lines based on radioligand binding and receptor internalization studies. Upon arginine vasopressin stimulation, this chimeric receptor induced robust calcium mobilization and increase of adenylate cyclase activity. The observed signaling activities are through the activation of the chimera instead of endogenously expressed receptors, as single amino acid changes in the second transmembrane regions of the chimera drastically reduced receptor efficacy and potency. Our results suggest that VRR1 has dual signaling properties in coupling to both G(q) and G(S) pathways. Analysis of native VRR1 receptor signaling pathway by using a recently identified ligand for VRR1 confirmed this conclusion and therefore validated the utility of the chimeric receptor approach for signaling pathway identification.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cell Line
  • DNA Primers
  • Fluorescent Antibody Technique
  • Humans
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Radioligand Assay
  • Receptors, Cell Surface / chemistry
  • Receptors, Cell Surface / metabolism*
  • Receptors, Vasopressin / chemistry
  • Receptors, Vasopressin / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism*
  • Sequence Homology, Amino Acid
  • Signal Transduction*

Substances

  • DNA Primers
  • Receptors, Cell Surface
  • Receptors, Vasopressin
  • Recombinant Fusion Proteins
  • vasopressin receptor-related receptor 1, human