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.

Abstract

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.

• Authors: Wen-Bo Zhang, Jean-Marc Navenot, Bodduluri Haribabu, Hirokazu Tamamura, Kenichi Hiramatu, Akane Omagari, Gang Pei, John P Manfredi, Nobutaka Fujii, James R Broach, Stephen C Peiper
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 277 Issue 27 Pg. 24515-21 (Jul 05 2002) ISSN: 0021-9258 [Print] United States
• PMID: 11923301 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Anti-HIV Agents
• Benzylamines
• Cyclams
• Heterocyclic Compounds
• N-alpha-acetyl-nona-D-arginine amide acetate
• Oligopeptides
• Receptors, CXCR4
• Recombinant Proteins
• T140 peptide
• GTP-Binding Proteins
• plerixafor

Topics

• Amino Acid Substitution
• Animals
• Anti-HIV Agents (pharmacology)
• Benzylamines
• CHO Cells
• Cricetinae
• Cyclams
• GTP-Binding Proteins (metabolism)
• Genes, Reporter
• Genetic Variation
• Heterocyclic Compounds (pharmacology)
• Humans
• Oligopeptides (pharmacology)
• Open Reading Frames
• Point Mutation
• Protein Conformation
• Receptors, CXCR4 (agonists, antagonists & inhibitors, genetics, metabolism)
• Recombinant Proteins (metabolism)
• Saccharomyces cerevisiae (drug effects, genetics, physiology)
• Signal Transduction
• Transfection