G protein coupling of the rat A1-adenosine receptor--partial purification of a protein which stabilizes the receptor-G protein association.
A membrane protein identified in cortical brain membranes and termed 'coupling cofactor', modulates G protein-coupling of the A1-adenosine receptor by reducing the catalytic efficiency of the receptor. Coupling cofactor traps the A1-adenosine receptor in the high affinity complex and, thus, is responsible for the resistance of high affinity A1-agonist binding to modulation by guanine nucleotides. In the present work, this effect was used for assaying the activity of coupling cofactor by reconstituting guanine-nucleotide resistant agonist binding to rat A1-adenosine receptors in detergent extracted brain membranes or in membranes from 293 cells after stable transfection with receptor cDNA. Coupling cofactor was partially purified from porcine brain membranes. The specific activity was modestly enriched (approximately 5-fold) after three chromatographic steps (DEAE-Sephacel, AcA34, MonoQ pH 8). Rechromatography of coupling cofactor over MonoQ at pH 7 resulted in a loss in specific activity if membranes of 293 cells but not if brain membranes were used as acceptor membranes. In addition, the molecular mass estimated by gel filtration decreased from > 150 kDa in the initial stage of purification to 40-30 kDa after this fourth chromatographic step. These two observations suggest that coupling cofactor requires an additional component that is present in brain membranes and is lost in later stages of purification. The activity of partially purified preparations of coupling cofactor activity relied also on the abundance of G protein alpha-subunits in the membrane. The activity on reconstitution with brain membranes or pertussis toxin pretreated 293 membranes was supported by addition of Gi alpha (rank order of protency: alpha i1 > alpha i3 > alpha i2) but not of G(o alpha). The selectivity for G protein alpha-subunits suggests that coupling cofactor may provide for an additional level of specificity in organizing receptor-G protein coupling.
Nanoff
C
C
Institute of Pharmacology, Vienna University, Austria. christian.nanoff@univie.ac.at
Waldhoer
M
M
Roka
F
F
Freissmuth
M
M
D000818:Animals; D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D018076:DNA, Complementary / Q000235:genetics; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D011869:Radioligand Assay; D051381:Rats; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D014162:Transfection
D018076:DNA, Complementary; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018047:Receptors, Purinergic P1; D019204:GTP-Binding Proteins