Parotid acinar cells, which are specialized for stimulus-dependent secretion of proteins, sort their secretory products intracellularly for either storage in secretion granules and stimulus-dependent exocytosis or constitutive release in a recently discovered non-granular pathway. Secretory sorting introduces a new factor in understanding the control of salivary composition, particularly during intervening periods between amplified salivary stimulation. The long range objectives of the proposed research are to document earlier implications that the maturing storage granule constitutes a site for this type of exocrine secretory sorting and to identify structural determinants on selected parotid salivary polypeptides that potentially serve as sorting signals.
The specific aims are organized around two lines of investigation. First, site(s) of divergence of the granule storage and constitutive pathways will be examined immunocytochemically using monospecific antibodies to structurally related 22 and 16 kDa secretory proteins that are differentially sorted between the two discharge routes. Subsequently, the primary structures of these polypeptides will be deduced by cloning and sequencing appropriate cDNAs isolated from a parotid cDNA library. This will enable further comparative analysis of higher order and expression experiments in other cell types exhibiting similar types of secretory pathways in order to check if differential sorting of these species is a general phenomenon. The second line of studies comprises a molecular dissection of one of the proline rich secretory proteins (PRP) in order to check whether linear structural information in these elongate and minimally folded macromolecules might govern their efficient storage in secretory granules. Using the cloned cDNAs for the PRP, a prokaryotic periplasmic protein (alkaline phosphatase), and genetically engineered fusions of PRP segments to alkaline phosphatase, expression studies in endocrine and exocrine cell lines will be conducted to evaluate, respectively, the extents of granule storage, constitutive secretion, and rerouting from constitutive secretion to granule storage.
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