The major objective of this proposal is to characterize cellular and subcellular mechanisms involved in the neuronal storage and release of serotonin (5-HT). Although found only in relatively small numbers of central and peripheral neurons, this neurotransmitter has major effect on behavior and mood. During the previous project period two specific serotonin (5-HT)) binding proteins (45 kDa and 56 kDa SBP) were purified and characterized. These proteins are found only in that subset of 5-HT-storing cells that is derived from neuroectoderm. It was proposed that 56 kDa SBP may be a precursor of the 45 kDa protein and that 45 kDa SBP may play a role in the storage of 5-HT in synaptic vesicles. These hypotheses will be tested in the proposed research (i) In order to determine if 56 kDa SBP is the physiological precursor of the 45 kDa molecule a determination will be made of (a) the extent of sequence homology between 56 kDa and 45 kDa SBP; (b) whether metabolically labeled 35S-56 kDa SBP is synthesized before 35S-45 kDa SBP and whether or not the 56 kDa protein is converted to the 45 kDa material. Monospecific antibodies to 45 kDa and 56 kDa SBP that were prepared during the prior project period will make these studies possible. This investigation will be facilitated by studying a recently characterized cell line (MTC cells) that produces and stores both 5-HT and SBP. If SBP is really an intravesicular 5-HT storage protein, it would be expected to be synthesized with a signal sequence, translated on polysomes attached to the RER, and cotranslationally segregated in the lumen of the RER. These predictions will be tested by in vitro translation using poly(A)+RNA extracted from the rat brain. The products (and their resistance to proteolysis) of in vitro translation with a rabbit reticulocyte lysate system will be compared with those synthesized on dog pancreas microsomes, in order to determine if cleavage of a signal peptide occurs and if there is vectorial transport of newly synthesized SBP into microsomes. If the initial experiments show that 56 kDa is produced before the 45 kDa SBP the intracellular compartment where the 56 kDa leads to 45 SBP conversion occurs, will be located. This will be done by following the fate, under control and experimental conditions, of pulse labeled SBP as it passes through successive subcellular compartments during a chase period. In additional experiments the ontogeny of serotonergic synapses will be studied. In particular, whether the timing of the appearance of 45 kDa SBP during ontogeny coincides with the development of serotonergic synaptic vesicles will be determined. Finally, the possibilities that 45 kDa SBP is co-released with 5-HT from activated serotonergic cells and is recaptured will be tested.
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