Pergamon PII: s0028-3908(9'r)00135-4 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Neuropharmacology , Vol. 36, No. 9, pp. 1211-1219, 1997 0 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 002%3908/97 $17.00 + 0.00 G Protein Coupling of the Rat Al-Adenosine Receptor- Partial Purification of a Protein Which Stabilizes the Receptor-G Protein Association CHRISTIAN NANOFF,* MARIA WALDHOER, FLORIAN ROKA and MICHAEL FREISSMUTH Institute of Pharmacology, Vienna University, Wiihringer Strape 13a, A-1090 Vienna, Austria (Accepted 27 June 1997) Summary-A membrane protein identified in cortical brain membranes and termed ‘coupling cofactor’, modulates G protein-coupling of the Al-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 Al -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 Al-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 (N 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 a-subunits in the membrane. The activity on reconstitution with brain membranes or pertussis toxin pretreated 293 membranes was supported by addition of Gi, (rank order of protency: ail > ai > aiz) but. not of Go,. The selectivity for G protein a-subunits suggests that coupling cofactor may provide for an additional level of specificity in organizing receptor-G protein coupling. 0 1997 Elsevier Science Ltd. Keywords-Al -adenosine receptor, G proteins, agonist binding, purification. The purine nucleoside adenosine is present in high nanomolar concentrations in the extracellular fluid of many tissues including the central nervous system. The adenosine levels in the brain rise rapidly during increased nerve activity, hypoglycaernia, hypoxia and ischaemia and extracellular adenosine is thought to be physiologi- cally important for neuroprotection (Rudolphi et al., 1992), for example, by antagonizing the excitotoxic effects of glutamate (Manzoni et al., 1994). Adenosine is a powerful inhibitor of neurotransmitter release from presynaptic nerve terminals. In addition, it modulates postsynaptic action potentials and causes hyperpolariza- tion of neuronal cells [for a review see Rudolphi et al. (1992)]. Both pre- and postsynaptic actions are mediated *To whom correspondence should be addressed. Tel.: 43-l-40 480/298; Fax: 43-l-402. 48 33; E-mail: christian.nanoff@ univie.ac.at. via Al-adenosine receptors which are widely distributed in the brain with the highest densities in cortex, hippocampus and cerebellum. The signal produced by Al-adenosine receptor activation is transduced to a G protein of the Gi/, class leading to the modulation of effecters, for example, enzymes such as adenylyl cyclase and phospholipase Cj5isoforms. In neuronal cells, Al- adenosine receptor stimulation has long been known to inhibit Ca2+ currents and activate potassium currents (Fredholm et al., 1994). Reconstitution studies with the Al-adenosine receptor purified from bovine brain or expressed in Escherichiu coli membranes adequately reproduced the basic char- acteristics of receptor G protein coupling and indicated a preference of the Al-adenosine receptor to associate with Gi, over G,,-subunits (Freissmuth et al., 1991b; Munshi et al., 1991; Jockers et al., 1994). The coupling between Al-adenosine receptor and G protein in rat brain 1211