The long-term objective is to elucidate the molecular mechanism of action of hormone receptors.
Specific aims are: I. To define the requirement for specific lipids in interaction of the Beta-adrenergic receptor (R) with the guanyl nucleotide binding protein (G). II. To further characterize the specific reversible locking of hormone (H) in R. III. To purify R so that it will retain its ability to activate and adenylate cyclase. IV. To characterize the action of new, high affinity, adrenergic agonists and to employ their labeled derivatives as monitors of active states of the receptor. V. To determine the relative amounts of R/G/C (catalytic unit of adenylate cyclase) in the cell membrane in terms of their activities. The work is health related in establishing criteria for analysis of normal and abnormal function of R and its interaction with G. Studying new, highly potent catecholamines should help in the development of new drugs. Methodology: I. Solubilized R and G will be delipidated on Ultrogel columns. After reconstitution with specific lipids, addition of hormone plus Gpp(NH)p should produce activated GGpp(NH)p. II. Addition of small amounts of deoxycholate to turkey erythrocyte membranes locks prebound [3H] isoproterenol in R. Other detergents, metal ions, etc. will be studied to characterize this induced fit of R to the agonist. III. A stable HRG complex will be prepared by alkylation of a unique -SH on G with a synthetic maleimide linked to biotin. The solubilized complex will be purified on an avidin column, and the receptor will be finally rescued by dissociation from the complex. IV. At the suggestion of Drs. Goodman and Melmon, new catecholamines which they originated will be studied with respect to binding and locking of the labeled compounds by Beta1 and Beta2 receptors. V. Near maximal activation of C will be performed by H + GTPGammaS to produce GGTPGammaS-C. Residual non-activated G available for locking R by H plus N-ethylmaleimide will then be determined. Transfer to an S-49 lymphoma variant which lacks functional G will determine the amount of activated and non-activated G.
