Restoration of glucagon responsive transformed rat hepatocytes (RL-P progenitor cells and rat liver plasm glucagon receptors/adenylate cyclase/polyethylene glycol)

Spontaneously transformed RL-PR-C rat hepa- tocytes, unlike their normal differentiated progenitor cells, are insensitive to glucagon, although seemingly possessing large num- bers of glucagon receptors and although retaining a guanyl nu- cleotide regulatory protein-adenylate cyclase (ATP pyrophos- phate-lyase (cyclizing), EC 4.6.1.1) system that responds to catecholamines, cholera toxin, and fluoride ions. Biochemical fu- sions between normal RL-PR-C hepatocytes or purified rat liver plasma membranes (whose adenylate cyclases were previously ir- reversibly inactivated with N-ethylmaleimide) with spontaneously transformed hepatocytes produced hybrids whose basal and flu- oride-stimulated adenylate cyclase activities reflected those of the parental transformed cells but that now responded to glucagon. Using cholera toxin-catalyzed ADP-ribosylation of transformed hepatocytes to mark their guanyl nucleotide regulatory protein, fusing such cells with N-ethylmaleimide-treated normal hepato- cytes, and examining glucagon stimulation of adenylate cyclase activity in fusion hybrids produced results suggesting that the reg- ulatory protein of the transformed cells is functionally normal. In fusion experiments between N-ethylmaleimide-treated hepato- cytes and pigeon erythrocytes, we found that normal, but not transformed, hepatocytes were effective in conferring glucagon sensitivity upon erythrocytes. Glucagon binding data revealed that, whereas normal RL-PR-C hepatocytes have two independent classes of binding sites, one of higher and the other of lower af- finity, transformed cells possess only the low-affinity receptors. From these and previous observations, it is possible to conclude that the insensitivity of spontaneously transformed RL-PR-C he- patocytes to glucagon is due to the loss, during the transformation process, of the high-affinity glucagon receptor.