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TxE CoUNCU. FOR ToBACCO RE81EARCS-U.S.A., INC. TO : DR. S. C. SOrIIMERS AND STAFF FROM: D. H. Ford zH~gal~ `~T" , Rockefeller University, RE: Site visit with Dr. R.' N.Y.C., March.18, 1986 Site visitors: D. H. Ford and H. McAllister Grant No. 1696 entitled "The Nicotinic Receptor: Regulation by Protein Phosphorylation" GOALS To explore the role of protein phosphorylation in the regulation of the function of the nicotinic acetyicholine receptor. RESULTS Dr. Huganir and colleagues have demonstrated that phosphorylation of the ACh receptor is catalized by at least three different protein kinases (a cAMP-dependent kinase, protein kinase C and a tyrosine-specific protein kinase) which may act on seven different phosphorylation sites. They have examined the functional effects of phosphorylation of the nACh receptor by cAMP-dependent protein kinase and found that the kinase phosphorylates thefand 6 subunits of the receptor, increasing the rate of rapid desensi- tization. This is a process whereby the receptor is inactivated by ACh, thus differing from the results which occur when ACh binds to the subunit, activating the receptor and opening the ion channel. Thus, the study provides evidence whereby a receptor made up of five subunits surrounding a channel pore can be activated (ion channel open) by the acti- vation of one subunit (y() and deactivated by a reaction at two of the other subunits (X-and 6). This information is contained in a recently published paper to Nature (ACKs CTR) entitled "Phosphorylation of the Nicotinic Acetylcholine Receptor Modulates its Rate of Desensitization," Huganir, et al. In another recent publication "Protein Phosphorylation and Neuronal Function," Browning et al, J:'Neurochem. 45:11, 1985, Huganir reviews the general role of phosphorylation in neuronal function. FUTURE DIRECTIONS Dr. Huganir indicated that their studies with Torpedo californicus nACH receptors were about concluded and that he will now be beginning to direct his investigations toward understanding the nASh receptor in the CNS. His studies will be directed toward determining what or how a second messenger system in neurons is-regulated; to what degree catecholamines and peptides influence the response of the ACh receptor, how responses of adjacent re- ceptors influence the response of the ACh receptor and how paracrine mediated influences may modify nACh receptor responses. The effects of catecholamines,

HUGANIR SITE VISIT GRANT #1696 Page 2 peptides and paracrine responses will be attempted first by using the ACh neuromuscular junction model. Another study to be undertaken is to de- termine by electronmicroscopic immunocytochemistry the localization of the tyrosine-specific protein kinase activity. COMMENT Dr. Huganir is a young, extremely bright and well-informed investi- gator who appears capable of examining ACh receptor function with an open mind in relation to a number of possible mechanisms which may act either directly or indirectly. At the time of our first visit we (Ford, McAllister and Stone) were somewhat concerned as to the degree by which his investiga- tions were directed or influenced by Dr. P. Greengaard, who directs his laboratory group. However, after this visit, both Dr. McAllister and I feel that the investigation Huganir is conducting represents essentially his own interest. His current work is both fresh and innovative and appears likely to contribute significantly to our understanding of the function of the nACh receptor and perhaps others wherein phosphorylation occurs. This is good work and merits continued support. D. H. F.