In vitro reconstitution and preparative purification of complexes between the chemokine receptor CXCR4 and its ligands SDF-1alpha, gp120-CD4 and AMD3100.
CXCR4 belongs to the family of G protein-coupled receptors and mediates the various developmental and regulatory effects of the chemokine SDF-1alpha. In addition, CXCR4 acts as a co-receptor along with CD4 for the HIV-1 viral glycoprotein gp120. Recently, there has also been a small molecule described that antagonizes both SDF-1 and gp120 binding to CXCR4. The structural and mechanistic basis for this dual recognition ability of CXCR4 is unknown largely due to the technical challenges of biochemically producing the components of the various complexes. We expressed the human CXCR4 receptor using a modified baculovirus expression vector that facilitates a single step antibody affinity purification of CXCR4 to >80% purity from Hi5 cells. The recombinant receptor undergoes N-linked glycosylation, tyrosine sulfation and is recognized by the 12G5 conformation specific antibody against human CXCR4. We are able to purify CXCR4 alone as well as complexed with its endogenous ligand SDF-1, its viral ligand gp120, and a small molecule antagonist AMD3100 by ion-exchange chromatography. We anticipate that the expression and purification scheme described in this paper will facilitate structure-function studies aimed at elucidating the molecular basis for CXCR4 recognition of its endogenous chemokine and viral ligands.
Dukkipati
Abhiram
A
Department of Microbiology and Immunology, Stanford University School of Medicine, 299 Campus Drive, Stanford, CA 94305, USA.
Vaclavikova
Jana
J
Waghray
Deepa
D
Garcia
K Christopher
KC
D000595:Amino Acid Sequence; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001596:Benzylamines; D015704:CD4 Antigens / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002478:Cells, Cultured; D054377:Chemokine CXCL12; D019743:Chemokines, CXC / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000080027:Cyclams; D019281:Dimerization; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006571:Heterocyclic Compounds / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D019718:Receptors, CXCR4 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D001596:Benzylamines; D015704:CD4 Antigens; C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D019743:Chemokines, CXC; D000080027:Cyclams; D015699:HIV Envelope Protein gp120; D006571:Heterocyclic Compounds; D019718:Receptors, CXCR4; D011994:Recombinant Proteins; C088327:plerixafor