SIGNAL TRANSDUCTION IN SALIVARY GLAND

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Acetylcholine, norepinephrine and substance P control salivary secretion by binding to receptors on the surface of salivary acinar cells to produce specific intracellular signals. Two signal transduction pathways have been identified: one involves cAMP synthesis and the other involves phosphoinositide hydrolysis and Ca2+ mobilization. The cAMP pathway, including the role of G proteins as transducers, is well understood. Less is known about the phosphoinositide /Ca2+ pathway although there is evidence that a G protein is also involved in this pathway. The identity of the G protein is unknown; its lack of sensitivity to cholera and pertussis toxins would seem to rule out the involvement of any of the G proteins that have been purified to date. The objectives of this proposal are to identify this novel G protein, provisionally termed G(p), and to characterize its biochemical and functional properties. The specific aims are: 1) isolation and biochemical characterization of a pertussis toxin-insensitive G protein(s) from submaxillary glands that can functionally interact with the SP receptor; 2) identification of the G protein associated with the SP receptor, by chemical crosslinking; 3) analysis of the functional interactions of purified G with the SP receptor and phospholipase C; 4) partial amino acid sequence analysis of homogeneous GP. Two new experimental approaches will be used. Salivary gland membranes, depleted of their endogenous G proteins by treatment with alkaline buffer, will be reconstituted with active G proteins obtained during chromatographic fractionation of G proteins from horse submaxillary gland. This convenient and sensitive assay detects G proteins that can functionally interact with SP receptors to restore GTP-sensitive, high affinity agonist binding The second approach will involve the use of a novel photoreactive analog of SP. (125)I-labelled Phe8(pBz)-SP can be photoincorporated efficiently into the G -coupled SP receptor. Gp will then be chemically crosslinked to the photolabelled SP receptor and the resulting radiolabelled covalent adduct subjected to compositional analysis by SDS-PAGE and immunodetection techniques. There are disease states that result from altered G protein function. Understanding the role of G in the phosphoinositide/Ca2+ pathway will permit an assessment of altered transduction in certain states of salivary gland dysfunction.