The alpha2 adrenergic receptor produces cellular responses through activating a guanine nucleotide binding protein (G protein). Very little is known about the initial interactions of agonists with these receptors and the protein conformational changes accompanying those interactions. We are studying the alpha2 adrenergic receptor as a model of agonist-receptor- G protein interactions in membranes. High affinity agonist radioligands permit direct measurements of agonist binding that can be correlated with receptor-mediated responses both in native membranes and in reconstituted systems. We will use rapid mix quench techniques to study the initial binding of [3H] UK 14,304, a full alpha2 agonist, on the 0.3 to 20 second time scale. Parallel rapid kinetic studies of adenylate cyclase inhibition in membranes and G protein activation in reconstituted systems will be carried out under identical conditions. The direct comparison between agonist binding and function will help identify the receptor states associated with G protein activation. In addition to the radioisotopic methods we will develop fluorescence spectroscopic methods to study the G protein conformational changes and subunit interactions induced by the alpha2 adrenergic receptor. For this purpose, G proteins labeled with extrinsic fluorophores will be prepared. Changes in fluorescence intensity will be used for real-time kinetic studies of G protein conformational changes in reconstituted systems. These probes will also be used for studies of fluorescence energy transfer and polarized photobleaching recovery. The latter two methods should provide insights into the validity of the G protein subunit dissociation model in a membrane system. The polarized photobleaching technique which we are developing in reconstituted systems will ultimately be applicable to studies of intact cells. This work will provide new information about the mechanism of alpha2 receptor activation by agonists. More generally, the information on receptor-G protein coupling is applicable to a wide range of biological signalling mechanisms including adenylate cyclase, inositol lipid metabolism and regulation of ion channels.
Showing the most recent 10 out of 29 publications
