The effect of palmitoylation on the function of the alpha subunits of the guanine nucleotide binding proteins G11 and G0

[摘要] Heterotrimeric guanine nucleotide binding proteins function to couple seven transmembrane spanning serpentine receptors to effector systems governing the regulation of intracellular second messengers. The basic molecular mechanism by which these G proteins operate has been known for several years, but the precise details of their functions, specificities and regulation is at present unresolved. These G proteins are characterised by being composed of three non-identical subunits termed alpha, beta and gamma. Molecular cloning techniques have revealed the presence of 17alpha, 4beta and 10gamma genes encoding these subunits. The alpha subunits have been classed into 4 groups termed the Gs, Gi, Gq and G12 families. Upon agonist occupation of a receptor, the G protein undergoes a conformational change causing GDP, which is bound to the alpha subunit in its inactive state, to be released and allowing GTP to enter the nucleotide binding pocket, causing release from the receptor and dissociation of the G protein into a free alpha subunit and a betagamma dimer. These moieties then interact with their target effector systems before the intrinsic GTPase activity of the alpha subunit hydrolyses the GTP to GDP, allowing heterotrimer reformation and receptor interaction, returning the system to its resting state. It was originally thought that the alpha subunit solely contained the sites for interaction with both the receptor and the effector. It is now apparent however that the betagamma dimer plays an important role in governing the specificity of G protein-receptor interaction, and can affect second messenger generating systems such as adenylyl cyclase, phosphoinositidase C and certain ion channels. G proteins are membrane associated proteins. The initial theory that the role of the betagamma complex was to anchor the alpha subunit to the membrane, via prenylated residues at the C terminus of the gamma subunit is largely outdated. The presence of the 14 carbon saturated fatty acid, myristic acid, at the N terminus of some Gi family members was thought to hold the key to this dilemma. Myristoylated peptides however do not have sufficient free energy to interact with the membrane. Also, not all myristoylated proteins are membrane associated and all of the other G proteins lack the necessary consensus sequence for N-myristoylation. Another mechanism must exist to account for the membrane interactions of G protein a subunits. ecently it has been demonstrated that the 16 carbon fatty acid, palmitic acid, is also present in the N terminal region of the majority of G protein a subunits, as well as several other proteins involved in intracellular signalling, notably Src family members. This lipidation is thought to be a post translational, reversible modification, generally occurring via labile thioester bonds. This is in contrast to N-myristoylation, which is co-translational and generally irreversible. This thesis aimed to determine the effect of mutating the target palmitoylation sites of rat G01alpha and murine G11alpha on their ability to interact with the plasma membrane when transfected into heterologous cell systems.(Abstract shortened by ProQuest.).

[发布机构] University:University of Glasgow

[关键词] Molecular biology