REGULATORY DOMAINS OF G-PROTEIN COUPLED RECEPTORS

G蛋白偶联受体是一个多基因细胞家族的成员

G protein-coupled receptors are members of a multigene family of cell surface receptors that regulate cellular responses to a variety of~extracellular signals including hormones, chemotactic agents and sensory stimuli. These events are important in the control of growth, differentiation and metabolism in eucaryotic cells. The activation of G proteins by these receptors results in the generation of multiple second messengers, stimulation of phosphorylation cascades and regulation of a number of ion channels. Defects in these receptors, their G proteins or effector enzymes have been implicated in the control of both benign and malignant tumor formation as well as a number of metabolic disorders. For example, naturally occurring point mutations in rhodopsin are responsible for several forms of the disease retinitis pigmentosa which results in a partial destruction of the mammalian retina causing blindness. Rhodopsin, the photoreceptor of the mammalian rod cell, has been used as a structural model for investigating the molecular interactions between G protein-coupled receptors and their G proteins and the process of receptor desensitization, which involves a loss of sensitivity of the receptor to its original stimulus. The primary focus of this research proposal is to utilize bovine rhodopsin as a model system for studying the molecular interactions between G protein-coupled receptors, G proteins and the kinases and arrestins involved in receptor desensitization. In order to achieve this goal, point mutations and deletions will be generated in the cDNA for bovine opsin by site-directed mutagenesis of specific structural domains predicted to be involved in these important signal transduction processes. This will be followed by transient expression of these altered proteins in HEK-293 cells for generating large quantities of the mutant opsin proteins and functional reconstitution with the rod cell G protein, Gt, as well as rhodopsin kinase, protein kinase C and arrestin. Domains in rhodopsin which are critical for the interaction of the photoreceptor with its G protein will be identified using light-dependent GTPgammaS binding to Gt. The effect of mutations on the observed cooperativity of the interaction between rhodopsin and its G protein will also be examined. The regulatory domains required for the light-dependent binding of rhodopsin kinase and arrestin will be identified by phosphorylation and binding assays. The serine/threonine-rich domain of the carboxy-terminus will be mutated to identify those sites that are most likely to be phosphorylated by rhodopsin kinase and protein kinase C and those that are most critical for the binding of arrestin. A more complete understanding of the mechanisms by which overlapping domains within rhodopsin control the processes of G protein activation and desensitization will aid in defining potential mechanisms of disease resulting from defects in G protein-coupled receptor signaling pathways.