A point mutation that confers constitutive activity to CXCR4 reveals that T140 is an inverse agonist and that AMD3100 and ALX40-4C are weak partial agonists.

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. To elucidate the mechanism for CXCR4 activation, a constitutively active mutant (CAM) was derived by coupling the receptor to the pheromone response pathway in yeast. Conversion of Asn-119 to Ser or Ala, but not Asp or Lys, conferred autonomous CXCR4 signaling in yeast and mammalian cells. SDF-1 induced signaling in variants with substitution of Asn-119 to Ser, Ala, or Asp, but not Lys. These variants had similar cell surface expression and binding affinity for SDF-1. CXCR4-CAMs were constitutively phosphorylated and present in cytosolic inclusions. Analysis of antagonists revealed that exposure to AMD3100 or ALX40-4C induced G protein activation by CXCR4 wild type, which was greater in the CAM, whereas T140 decreased autonomous signaling. The affinity of AMD3100 and ALX40-4C binding to 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 of a new generation of agents that lack partial agonist activity that may induce toxicities, as observed for AMD3100.