The objective of the proposed research is to determine the nature, interrelationship, and relevance of lipid modifications to the anchorage and function of GTP-binding regulatory protein (G protein) alpha subunits. G proteins are integral to pathways of signal transduction established for a wide variety of hormones. The alpha subunit is the entity with which the identity of a G protein is most often equated, and is a major determinant in stipulating interactions of the G protein with receptors and target enzymes or channels. The various alpha subunits so far identified are subject to N-myristoylation and/or palmitoylation. The relevance of these modification is most often described in terms of anchorage to membranes. Despite the high level of interest in fatty acid acylation, however, basic issues of mechanism and functional relevance are poorly documented. The questions to be addressed in this proposal deal specifically with lipid modifications in the context of biosynthetic anchorage, subunit protein interactions, and subunit stability. The first goal of the project is to define elements of structure required for palmitoylation of G protein alpha subunits. The hypotheses to be tested are i) that N- myristoylation and/or anchorage is required for palmitoylation of alpha/i- like subunits, and ii) that a still undefined modification, or peptide sequence, serves a similar purpose in the palmitoylation of alpha/s-1, alpha/q-, and alpha/12-like subunits. Achievement of this goal will strengthen the concept that interactions with membranes are a prerequisite to palmitoylation, provide the framework for understanding why at least two different events exist to achieve palmitoylation, and provide the information necessary for identifying alternate modifications and/or points of membrane contact. The second goal of the project will be to determine whether hormonal regulation of G protein alpha subunit palmitoylation extends beyond that established for alpha/s, and can therefore serve as an index of G protein activation. The information to be obtained has not only mechanistic, but pragmatic value, since intact- cell assays of G protein activation have proven elusive. The third goal will be to quantitate the stoichiometry and heterogeneity of fatty acid acylation by mass spectrometry. This will provide quantitative insight into the dynamics of fatty acid acylation previously lacking in studies involving radioisotopic labeling. Mass spectrometry will additionally be used to explore other modifications. Finally, the fourth goal will be to investigate the relevance of palmitoylation to alpha subunit function. Hypotheses to be tested are i) that palmitoylation is normally redundant to other mechanisms of membrane attachment but assumes importance as these are disengaged, ii) that palmitoylation alone or together with N- myristoylation facilitates interactions with other proteins, e.g., receptors, beta gamma, and glycosylphosphatidylinositol-anchored proteins, and iii) that palmitoylation helps prevent degradation of the subunit in the intact cell.
