Photo affinity labelling of β-adrenergic receptors of C6 glioma cells☆Presence of a nucleophilic group in the receptor

Abstract (±) 1-[1-( p -Nitrophenoxy), 2-methyl, 2-propylamino], 3-(α-naphtyloxy), 2-propanol (PNP) was synthesized and found to be a potent photoaffinity label for β-adrenergic receptors of C 6 glioma cells. In the dark, PNP displaced all the [ 3 H]DHA binding sites on C 6 glioma cell membranes ( K D = 5.5 × 10 −8 M). Upon photolysis on isolated C 6 glioma cell membranes: (a) PNP reduced in a dose-dependent manner maximally stimulated β-adrenergic sensitive adenylate cyclase. After extensive washing of the membranes, the maximal β-adrenergic stimulation was reduced without change in the apparent affinity for isoproterenol. A 62% decrease in activity was obtained with 10 −5 M PNP without any change in basal and NaF stimulated adenylate cyclase activities, (b) PNP also irreversibly reduced in a dose-dependent manner the total number of [ 3 H]DHA binding sites without changing the affinity of the remaining sites. The effects of PNP on adenylate cyclase and [ 3 H]DHA binding were suppressed in the presence of (−)alprenolol. Upon photolysis on intact C 6 glioma cells, PNP inactivated β-adrenergic receptors coupled with adenylate cyclase without any change in basal, NaF and Gpp(NH)p stimulated adenylate cyclase activities. These results indicate that PNP photolabelling occurred on the β-adrenergic receptors. Furthermore, as PNP was shown to react with model nucleophiles upon photolysis, this labelling implies the presence of a nucleophilic group in the β-adrenergic receptor.