The proposed research is a direct continuation of an ongoing, NSF supported project, using the larvae of Haliotis rufescens (gastropod mollusc) as a model for experimental analysis of the mechanisms by which cells and organisms recognize and transduce exogenous chemical signals. With previous support it has been determined that these larvae require the receptor-mediated recognition of an exogenous GABA-mimetic oligopeptide (or GABA analogs) to induce metamorphosis; that the receptors (R1) involved in this signal-recognition are located on cilia that can be conveniently purified in quantities sufficient for biochemical analyses; that these receptors are subject to a ligand- induced down-regulation; and that the receptor-mediated signal is transduced by a cascade involving adenyl cyclase, activation of a protein kinase, and the opening of membrane channels for chloride ions. It has also been found that responsiveness to R1 receptor activation can be amplified 1,000-fold by a second group of signal molecules (diamino acids) that bind to independent receptors (R2) also present on the purified cilia. This signal amplification that results from R2 receptor binding is transduced by a separate pathway involving a G protein, phospholipase, diacylglycerol and a separate protein kinase. The next objectives of this study are to characterize further the cellular and molecular mechanisms of action of the signal receptors, transducers, and the amplifier described above. Specifically, it is proposed to: (1) purify (by affinity-techniques) the R1 and R2 receptors, the receptor- bearing cells and/or cilia, and the essential signal transduction components (including the targets of protein phosphorylation) of both the morphogenetic and amplifier pathways; (2) analyze the mechanisms of R1 and R2 receptor interaction with, and responses to, the cognate signal molecules in vitro; (3) resolve, reconstitute, and analyze in vitro the receptors and signal transduction elements essential for the function of both pathways, to determine their mechanisms of action at the molecular level; and (4) determine the molecular mechanism and the cellular level of interaction (i.e., signal amplification) of the morphogenetic and amplifier pathways, through analyses to be performed both in vitro and in vivo. This research will provide the most complete picture yet available of the molecular and cellular mechanisms by which chemical signals from the environment are recognized and transduced, and the mechanisms by which these processes are regulated, to contol the metamorphosis of marine invertebrate larvae. The results of these investigations also will provide new, basic information on the mechanisms of action and evolution of chemical signal receptors and their associated signal transducers and regulatory pathways, as well as the mechanisms by which these control physiological and behavioral processes, and responsiveness to stimuli. This information should be applicable to a wide variety of hormonal and other signal- and receptor-dependent systems.
