MOLECULAR ANALYSIS OF G PROTEIN ALPHA SUBUNIT FUNCTIONS

点击翻译按钮获取中文摘要

The heterotrimeric (alpha, beta, gamma) G proteins, a family of GTPases, transmit hormonal and sensory signals received by cell surface receptors to effector proteins that generate cellular responses. G proteins become activated when receptors catalyze the replacement of GDP bound to the alpha subunit with GTP. Alpha subunits share a high degree of similarity in their amino acid sequences. However, the differences among alpha subunits play a critical role in determining the specificity and nature of the interactions between receptors, G proteins, and effectors. Aberrant or subunits can cause pituitary, adrenal cortical, thyroid, and ovarian tumors, as well as McCune-Albright syndrome, type-1 pseudohypoparathyroidism, whooping cough, and cholera. The overall goal of the proposed studies is to understand the molecular mechanisms by which G protein alpha subunits transmit signals. Mutant alpha subunits will be generated and characterized biochemically to identify residues that specify interactions with particular receptors and effectors. These studies will be interpreted in the context of the recently solved X-ray crystal structure of the alpha subunit of transducin, the G protein that mediates vision. Combining structural and functional data will enable the development of a detailed molecular model of the mechanism of signal transduction by G proteins. Determining the requirements for productive interactions between receptors, alpha subunits, and effectors may lead to the rational design of therapeutic agents for use in treating diseases caused by aberrant G protein signaling pathways. The Specific Aims of this project are: (I) To establish how alpha-s interacts with the beta-adrenergic receptor. Mutant alpha-s constructs will be expressed and characterized in cyc S49 lymphoma cells, which are genetically deficient in alpha-s, to determine which alpha-s residues specify receptor interactions. To elucidate how alpha subunits transmit signals between receptors and effectors, the receptor-interacting surface defined by these residues will be related to the guanine nucleotide binding site, the alpha subunit regions that change conformation in response to GTP binding, and the previously identified adenylyl cyclase-activating surface of alpha-s. (II) To identify the residues of alpha-q that interact with phospholipase C (PLC). Mutant alpha-q constructs will be transiently expressed and characterized in a human embryonic kidney fibroblast cell line (HEK-293) to determine which alpha-q residues specify PLC interaction. PLC differs from adenylyl cyclase in that it is not an integral membrane protein. Furthermore, unlike adenylyl cyclase, PLC can activate the GTPase activity of the alpha subunit that stimulates it. Therefore, comparing the PLC- interacting surface of alpha-q with the adenylyl cyclase-activating surface of alpha-s will reveal the extent to which structurally conserved alpha subunits use common mechanisms to interact with diverse effectors.