My laboratory is interested in the molecular basis of cellular interactions during development. During the past 17 years, we have used a variety of biochemical, immunological, genetic and molecular biological approaches to show that beta1, 4-galactosyltransferase (GalTase) is present on the cell surface, in addition to its more traditional biosynthetic location in the Golgi complex. Cell surface GalTase functions as a cell adhesion molecule by binding to its complementary glycoconjugate substrate, or ligand , on adjacent cell surfaces and/or in the extracellular matrix. The purpose of this competitive renewal application is to continue our analysis of cell surface GalTase expression and function at the molecular level. During the previous funding period, we defined the role of surface GalTase during late morula compaction and F9 EC cell adhesion. Partial cDNAs encoding murine GalTase were cloned and used to clarify the confusion in the literature regarding multiple unrelated GalTase cDNAs. These partial clones were used to isolated the full-length cDNAs encoding the two different isoforms of GalTase, the """"""""long"""""""" and """"""""short"""""""" forms, which from two different in-frame ATGs, producing proteins that differ by a 13 amino acid sequence in the cytoplasmic domain of the """"""""long"""""""" protein. Using these cDNAs, the expression and subcellular distribution of GalTase during F9 cell differentiation was analyzed. A model was developed suggesting that a portion of the long GalTase is transported to the cell surface where it functions as a cell adhesion molecule, whereas the short GalTase serves a purely biosynthetic function in the Golgi. This model was confirmed by immunofluorescence confocal microscopy of the long GalTase in untransfected cells, by the production of a dominant negative mutation in surface GalTase function, and by producing a murine GalTase-dependent adhesive phenotype in human cells. In additional studies we examined the effects of altering GalTase expression on glycoprotein biosynthesis in F9 cells, defined the predominant cell surface ligands, or substrates, for surface GalTase on wild-type and adhesive-defective F9 cells, showed the GalTase elicits a growth-controlling and differentiation-inducing signal in F9 cells, showed that altering the expression of surface GalTase has a direct effect on cell growth rate, mapped genetic loci within the T/t-complex that regulate surface GalTase expression during spermatogenesis, examined GalTase's role in t-sperm transmission ratio distortion, and examined the effects of overexpressing sperm surface GalTase on fertilization in transgenic animals. These studies lead to the next seven Specific Aims that we will pursue during the coming funding period, which fall into two categories; 1) to assess the requirement of surface GalTase during fertilization and embryonic morphogenesis in vivo and cell interactions in vitro, and 2) to define the molecular basis of GalTase transport to the cell surface and association with signal transduction cascades.
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