G Protein-coupled Receptor Structure And Signal Transduction

CXCR4 is a G protein-coupled receptor for stromal-derived factor 1 (SDF-1)that plays a critical role in leukocyte trafficking, metastasis of mammary carcinoma,and human immunodeficiency virus type-1 infection. In order to facilitate the study ofits molecular structure, CXCR4 had been expressed in Saccharomyces cerevisiaelinked to the α-factor signal transduction pathway with a hybrid Gα protein subunit(yeast-Gpa1 and human Giα2) for the first time. SDF-1 induced dose-dependenthistidine prototrophy or, in strains also carrying a pheromone-responsive FUS1-lacZreporter gene, β-galactosidase expression. CXCR4 antagonists, including T22, T140,AMD3100, and vMIP-II, blocked the functions induced by SDF-1. Biochemicalanalysis revealed that CXCR4 underwent N-linked glycosylation in the yeast cellsand it had similar binding affinity to T140 as that expressed in mammalian cells. Aconstrained peptide corresponding to the third hypervariable (V3) domain of the IIIBenvelope glycoprotein blocked SDF-1-induced reporter gene expression in lowmicromolar concentrations. The functional expression of CXCR4 in this systemshould provide access to powerful genetic approaches in yeast that will provideinsight into the structural basis for the interaction of this protein with its cognateligand, antagonists, and HIV-1 envelope glycoproteins.To elucidate the mechanism for CXCR4 activation, a constitutively activemutant (CAM) was derived by using this system combined with random mutagenesis.Conversion of Asn-119 to Ser or Ala, but not Asp or Lys, conferred autonomousCXCR4 signaling in yeast and mammalian cells. SDF-1 induced signaling in variantswith substitution of Asn-119 to Ser, Ala, or Asp, but not Lys. These variants hadsimilar cell surface expression and binding affinity for SDF-1. CXCR4-CAMs hadhigh basal level phosphorylation and was present in cytosolic inclusions. Analysis ofantagonists revealed that exposure to AMD3100 or ALX40-4C induced G proteinactivation by CXCR4 wild type, which was greater in the CAM, whereas T140decreased autonomous signaling. The affinity of AMD3100 and ALX40-4C bindingto CAMs was less than to wild type, providing evidence of a conformational shift.These results illustrate the importance of transmembrane helix 3 in CXCR4 signaling.Insight into the mechanism for CXCR4 antagonists will allow for the development ofa new generation of agents that lack partial agonist activity that may induce toxicities,as observed for AMD3100.