Investigatiions on the functional regulation in the process of processing from the membrane-integrated form of HGF activator inhibitors

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Hepatocyte growth factor activator inhibitors type 1 (HAI-1) and type 2 (HAI-2) are Kunitz-type serine protease inhibitors, identified as strong inhibitors toward hepatocyte growth factor (HGF) activator. Each HAI molecule possesses the hydrophobic region at the C-terminus, suggesting that they are produced primarily in a membrane-integrated form with two Kunitz domains in its extracellular region, and ectodomain shedding releases several secreted forms. In order to clarify the physiological role of these regulatory factors, we have performed the functional analyzes of HAI-1 and HAI-2.1. Immunoblotting analysis revealed that HAI-1 is first produced in a 66 kDa membrane-integrated form, and subsequent ectodomain shedding releases two major secreted forms from the cell surface into the extracellular space, their sizes being 40/39 kDa and 58 kDa. Whereas the former containing one Kunitz domain shows strong inhibitory activity against the HGF-converting activity of HGF activator, the latte … More r containing two Kunitz domains shows markedly weaker inhibitory activity. Thus, the presence of the C-terminal Kunitz domain (Kunitz II) in the 58 kDa HAI-1 may interfere with the binding of HGF activator to the reactive site of the N-terminal Kunitz domain (Kunitz I), while elimination of this region by proteolytic processing could lead to strong binding of HGF activator to HAI-1.2. To determine roles of the Kunitz domains in the inhibitory activity of HAIs against serine proteases, we constructed various HAI mutants and examined their inhibitory activity against HGF activator. Kunitz I in each HAI molecule appears to be the functional domain for inhibiting the HGF-converting activity of HGF activator. Furthermore, the presence of two Kunitz domains affected the inhibitory activity of HAI-1 against HGF activator. These results suggest that serine protease binding sites of Kunitz I and Kunitz II are located close to each other, and proteolytic processing to generate HAI-1 with only one Kunitz domain regulates the activity of HAI-1. Identification of cognated serine proteases against Kunitz II will reveal the novel biological function of HAIs.3. In order to search for HAI secretases, we have examined protein-protein interaction in the cytoplasmic regions of HAIs using a yeast two-hybrid screening system. Several cytoplasmic proteins, including cytosleletal proteins and intracelluiar enzymes were obtained. One of them was known to be involved in suppression on apoptosis, suggesting that HAI may play important role on the regulation of death signaling (manuscripts in preparation).Further analyzes on the functional regulation in the process of the proteolytic processing of HAIs will provide a novel mechanism of the regulation on HGF activation. Less