THE AMPHIPHILIC PEPTIDE ADENOREGULIN ENHANCES AGONIST BINDING TO A(1)-ADENOSINE RECEPTORS AND [S-35] GTP-GAMMA-S TO BRAIN MEMBRANES

1. Adenoregulin is an amphilic peptide isolated from skin mucus of the tree frog, Phyllomedusa bicolor. Synthetic adenoregulin enhanced the binding of agonists to several G-protein-coupled receptors in rat brai n membranes. 2. The maximal enhancement of agonist binding, and in par entheses, the concentration of adenoregulin affording maximal enhancem ent were as follows: 60% (20 mu M) for A(1)-adenosine receptors, 30% ( 100 mu M) for A(2a)-adenosine receptors, 20% (2 mu M) for alpha(2)-adr energic receptors, and 30% (10 mu M) for 5HT(1A) receptors. High affin ity agonist binding for A(1-), alpha(2-), and 5HT(1A)-receptors was vi rtually abolished by GTP gamma S in the presence of adenoregulin, but was only partially abolished in its absence. Magnesium ions increased the binding of agonists to receptors and reduced the enhancement elici ted by adenoregulin. 3. The effect of adenoregulin on binding of N-6-c yclohexyladenosine ([H-3]CHA) to A(1)-receptors was relatively slow an d was irreversible. Adenoregulin increased the B-max value for [H-3]CH A binding sites, and the proportion of high affinity states, and slowe d the rate of [H-3]CHA dissociation. Binding of the A(1)-selective ant agonist, [H-3]DPCPX, was maximally enhanced by only 13% at 2 mu M aden oregulin. Basal and A(1)-adenosine receptor-stimulated binding of [S-3 5]GTP gamma S were maximally enhanced 45% and 23%, respectively, by 50 mu M adenoregulin. In CHAPS-solubilized membranes from rat cortex, th e binding of both [H-3]CHA and [H-3]DPCPX were enhanced by adenoreguli n. Binding of [H-3]CHA to membranes from DDT1 MF-2 cells was maximally enhanced 17% at 20 mu M adenoregulin. In intact DDT1 MF-2 cells, 20 m u M adenoregulin did not potentiate the inhibition of cyclic AMP accum ulation mediated via the adenosine A(1) receptor. 4. It is proposed th at adenoregulin enhances agonist binding through a mechanism involving enhancement of guanyl nucleotide exchange at G-proteins, resulting in a conversion of receptors into a high affinity state complexed with g uanyl nucleotide-free G-protein.