Cell membrane hybrid bilayers containing the G-protein-coupled receptor CCR5.
A hybrid bilayer membrane is a planar model membrane that is formed at an alkanethiol monolayer-coated gold surface by the spontaneous reorganization of phospholipid vesicles. Membrane vesicles from monkey kidney COS-1 cells also reorganize at an alkanethiol/lipid monolayer-coated surface resulting in the formation of a cell membrane hybrid bilayer. Atomic force microscopy and spectroscopic ellipsometry indicate that the cell membrane layer is equivalent to the thickness of one leaflet of the membrane and is continuous over large areas. Cell membrane hybrid bilayers were formed from membrane vesicles from COS-1 cells that were transiently transfected with a synthetic human CCR5 chemokine receptor gene. Preparations that contained "inside out" and "right side out" membrane vesicles were used. Binding of monoclonal antibodies to either the amino- or carboxyl-terminus of CCR5 was observed by surface plasmon resonance and confirmed the presence and the random orientation of these integral membrane receptors. Specific and concentration-dependent binding of the beta-chemokine RANTES to the cell membrane hybrid confirmed that CCR5 retained ligand-binding activity. The ability to form cell membrane hybrid bilayers that contain functional G-protein-coupled or other multispanning receptors without requiring protein isolation, purification, and reconstitution offers a promising method for the rapid screening of potential ligands.
Center for Cellular and Molecular Biology, Hyderabad, India.
N Madhusudhana
NM
Rao
Vitali
V
Silin
Kevin D
KD
Ridge
John T
JT
Woodward
Anne L
AL
Plant
D000818:Animals; D019556:COS Cells; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry; D018946:Chemokine CCL5 / Q000378:metabolism; D002678:Chimera; D002522:Chlorocebus aethiops; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D011485:Protein Binding; D019713:Receptors, CCR5 / Q000235:genetics / Q000378:metabolism*; D012243:Rhodopsin / Q000276:immunology; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance
D018946:Chemokine CCL5; D007074:Immunoglobulin G; D008024:Ligands; D008051:Lipid Bilayers; D008081:Liposomes; D008563:Membrane Lipids; D019713:Receptors, CCR5; D013438:Sulfhydryl Compounds; D012243:Rhodopsin