This proposal is based on our finding that cultured normal human foreskin cells synthesize and release into their medium three novel proteins (nexins) which specifically link certain regulatory serine proteases and mediate their binding to cells. The first nexin we found (Mr 38,000) links thrombin, urokinase or acrosin and perhaps the gamma subunit of nerve growth factor. The second nexin (Mr 95,000) specifically links the carrier protein for epidermal growth factor (EGF) while the third nexin (Mr 26,000) links the gamma subunit of nerve growth factor. These linked complexes then bind to cells apparently via the nexin moiety of the complex. We plan to purify the three nexins to homogeneity from serum-free conditioned medium and obtain polyclonal antibodies to each of them. This will enable us to develop radioimmunoassays for each nexin and accurately measure nexin release by cells. Using a variety of 125 Iota labeled regulatory proteases we will determine whether cells make nexins for proteases we have not yet tested and determine if these nexins are different from or identical to the three nexins identified so far. We will determine the relative linkage """"""""affinities"""""""" of the proteases to each purified nexin so it will be possible to assign the """"""""primary"""""""" protease(s) for each nexin. The radioimmunoassays will enable us to study the control of nexin biosynthesis and release by various proteases, the nexins themselves and certain other agents and determine if this control is incividual or coordinate for the nexins. We will prepare partial peptide maps of the nexins to determine their degree of relatedness and whether a fragment of each is removed during formation of nexin: protease complexes. With the availability of purified nexins, it will be possible to answer many critical questions concerning the interactions of the proteases, the nexins, and of nexin: proteases complexes with cells and to evaluate the consequences of these interactions. Finally, we will determine if the EGF carrier protein and its nexin control delivery of free EGF carrier protein and its nexin control delivery of free EGF to cells as suggested by preliminary studies.
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