Reconstitution and characterization of the human neutrophil N-formyl peptide receptor and GTP binding proteins in phospholipid vesicles.
We have developed a unilamellar phospholipid vesicle system which contains the N-formyl peptide receptor and GTP binding proteins. Several detergents were investigated but only two, octyl glucoside (35 mM) and deoxycholate (7.5 mM), were capable of extracting N-formyl peptide receptor from neutrophil membranes in a form which remained functionally active upon reconstitution into phospholipid vesicles. Extracted proteins were reconstituted into phosphatidylcholine vesicles by passage over a Sephadex G-50-80 column. The reconstituted formylpeptide receptor could bind [3H]FMLP (3H-labeled fMet-Leu-Phe) and [125I]FMLPL-SASD (125I-labeled N-formylmethionylleucylphenylalanyl-N epsilon-(2-(p-azidosalicylamido)ethyl- 1,3'-dithiopropionyl)lysine) while the endogenous G protein could bind [35S]GTP gamma S. Furthermore, the functional interaction of the two proteins was preserved. Addition of the nonhydrolyzable guanine nucleotide, GTP gamma S, shifted the N-formyl peptide receptor from a high- to a low-affinity binding state for ligand. The development of this in vitro reconstitution system should provide a basis to study the mechanism of interaction of the N-formyl peptide receptor and the G protein.
Cupo
J F
JF
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, CA.
Allen
R A
RA
Jesaitis
A J
AJ
Bokoch
G M
GM
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D015153:Blotting, Western; D002462:Cell Membrane / Q000032:analysis; D003902:Detergents; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000502:physiology; D006801:Humans; D008854:Microscopy, Electron; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000502:physiology*; D009504:Neutrophils / Q000648:ultrastructure*; D010743:Phospholipids; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000302:isolation & purification / Q000502:physiology
D003902:Detergents; D010743:Phospholipids; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D000246:Adenosine Diphosphate Ribose; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019204:GTP-Binding Proteins