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.
Zhang
Wen-Bo
WB
Henry Vogt Cancer Research Institute, University of Louisville, Louisville, Kentucky 40202, USA.
Navenot
Jean-Marc
JM
Haribabu
Bodduluri
B
Tamamura
Hirokazu
H
Hiramatu
Kenichi
K
Omagari
Akane
A
Pei
Gang
G
Manfredi
John P
JP
Fujii
Nobutaka
N
Broach
James R
JR
Peiper
Stephen C
SC
D019943:Amino Acid Substitution; D000818:Animals; D019380:Anti-HIV Agents / Q000494:pharmacology; D001596:Benzylamines; D016466:CHO Cells; D006224:Cricetinae; D000080027:Cyclams; D019204:GTP-Binding Proteins / Q000378:metabolism; D017930:Genes, Reporter; D014644:Genetic Variation; D006571:Heterocyclic Compounds / Q000494:pharmacology*; D006801:Humans; D009842:Oligopeptides / Q000494:pharmacology*; D016366:Open Reading Frames; D017354:Point Mutation; D011487:Protein Conformation; D019718:Receptors, CXCR4 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction; D014162:Transfection
D019380:Anti-HIV Agents; D001596:Benzylamines; D000080027:Cyclams; D006571:Heterocyclic Compounds; C094365:N-alpha-acetyl-nona-D-arginine amide acetate; D009842:Oligopeptides; D019718:Receptors, CXCR4; D011994:Recombinant Proteins; C116977:T140 peptide; D019204:GTP-Binding Proteins; C088327:plerixafor