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The motility of gastrointestinal tract is regulated by classical neurotransmitters, neuropeptides, and humoral agents. Two novel human cDNAs have been cloned based on their sequence similarity to a frog skin secretion protein, Bv8, and a nontoxic protein of mamba snake venom. These human cDNAs encode two secreted proteins of 86 and 81 amino acids. Northern blot hybridization has revealed that these cDNAs are expressed in gastrointestinal tract, particularly the stomach. Recombinant proteins with authentic N-terminal sequences have been produced in Escherichia coli and refolded into functional proteins by careful control of protein aggregation. Mass spectrometry has confirmed the formation of five pairs of disulfide bonds. The refolded recombinant proteins potently contract gastrointestinal smooth muscle with EC(50) values in the subnanomolar range. The contractile effects of the recombinant proteins are specific for gastrointestinal smooth muscle, because they have no effect on vascular or respiratory smooth muscle. To reflect their potent and specific effects on gastrointestinal smooth muscle cells, we have named these recombinant proteins prokineticins. Ligand binding studies with iodinated prokineticin revealed the presence of a high-affinity site in ileal smooth muscle. The displacement of specific binding by GTP gamma S suggests that the prokineticin receptor may belong to the family of G protein-coupled receptors. Experiments with verapamil and nifedipine revealed that calcium influx is essential for the contractile activity of prokineticins on gastrointestinal smooth muscle. In summary, we have identified two novel endogenous regulators of gastrointestinal motility. The availability of recombinant prokineticins should provide novel therapeutic agents for disorders involving impaired gastrointestinal motility.

We recently identified a synthetic peptide, Pro47-Ile58, derived from the mature protease nexin 1 (PN1) sequence, that inhibited the low density lipoprotein receptor-related protein (LRP)-mediated internalization of thrombin-PN1 (Th-PN1) complexes. Presently, we have analyzed this sequence in Th-PN1 complex catabolism using two independent approaches: 1) An antibody was generated against Pro47-Ile58, which inhibited complex degradation by 70% but had no effect on the binding of the complexes to cell surface heparins. This places the structural determinant in PN1 mediating complex internalization by the LRP outside of the heparin-binding site. 2) Site-directed genetic variants of PN1 with a single Ala substitution at His48, or two Ala substitutions, one at His48 and another at Asp49, were expressed in Sf9 insect cells. The catabolic rate of complexes formed between Th and the singly substituted and doubly substituted variants was lowered to 50 and 15%, respectively, when compared with the catabolic rate of native Th-PN1 complexes. This is the first analysis of a structural determinant in a serine protease inhibitor (SERPIN) required for LRP-mediated internalization and in part may explain the cryptic nature of this site in the unreacted serine protease inhibitor.

Protease nexin 1 (PN1) is a serine protease inhibitor (SERPIN) that acts as a suicide substrate for thrombin (Th) and urokinase-type plasminogen activator (uPA). PN1 forms 1:1 stoichiometric complexes with these proteases, which are then rapidly bound, internalized, and degraded. The low density lipoprotein receptor-related protein (LRP) is the receptor responsible for the internalization of protease-PN1 complexes. However, we found that the LRP is not significantly involved in the initial cell surface binding of thrombin-PN1, leading us to investigate what cellular component was responsible for this initial interaction. Since Th-PN1 complexes retain a high-affinity for heparin after complex formation, unlike several of the other SERPINs, we tested the possibility that cell surface heparins were involved in initial complex binding. Soluble heparin was found to be a potent inhibitor of the binding of Th-PN1 to the cell surface and greatly facilitated the dissociation of Th-PN1 complexes pre-bound in the absence of soluble heparin. To ascertain the role of cell surface heparins, further studies were done using complexes of thrombin and PN1(K7E), a variant of PN1 in which the heparin binding site was rendered non-functional. When added at equal initial concentrations of complexes, Th-PN1(K7E) was catabolized 5- to 10-fold less efficiently than Th-PN1, a direct result of the greatly diminished initial binding of the Th-PN1(K7E) complexes. These data demonstrate the sizable contribution of cell surface heparins to Thrombin-PN1 complex binding and support a model in which these heparins act to concentrate the complexes at the cell surface facilitating their subsequent LRP-dependent endocytosis.

Multiplication-stimulating activity (MSA) produced by Buffalo rat liver cells (BRL-3A) in culture is related to the somatomedin family of growth regulatory polypeptides. MSA will stimulate glucose transport and DNA synthesis in normal chicken embryo fibroblasts (CEF) at concentrations of 10-200 ng/ml. MSA found in BRL-3A-conditioned medium, like the somatomedins in serum, does not exist as the free hormone but is bound to a specific high molecular weight carrier protein. In this report we demonstrate that purified MSA carrier protein (MCP) inhibits the biological activity of MSA on CEF as measured by the stimulation of glucose transport and DNA synthesis. In addition, purified MCP competitively inhibits the binding of 125I-labeled MSA to these cells. In control experiments in which insulin was used as the mitogenic agent, MCP had no effect on these biological responses. These results indicate that the inhibitory effect of MCP is the result of specific interaction with MSA and support the hypothesis that cells may be unresponsive to somatomedins bound to their serum carrier proteins.

An overlapping synthetic peptide library was constructed representing most of the mature protease nexin I (PN1) sequence from the amino terminus to the reactive center. This library, along with peptides from the heparin binding domain and from the region carboxyl-terminal to the P1 residue of the cleavage site, was screened for the inhibition of 125I-thrombin (Th)-PN1 complex binding and degradation. A peptide corresponding to residues Pro47-Ile58 in the PN1 sequence was identified as a potent inhibitor of 125I-Th-PN1 complex degradation, although it did not affect binding significantly. Pro47-Ile58 was shown to competitively inhibit the low density lipoprotein receptor-related protein (LRP)/alpha2-macroglobulin receptor-mediated endocytosis of 125I-Th-PN1 complexes in mouse embryo fibroblasts. Pro47-Ile58 is an apparent transition sequence in PN1, separating sheet-6B and helix-B. The sequence of Pro47-Ile58, PHDNIVISPHGI, suggests that it forms a loop structure defined by the seven underlined amino acids bordered by proline residues at each end. These studies are the first to identify a putative binding site in a serine protease inhibitor that is required for LRP-mediated internalization.

Recombinant native antithrombin III (ATIII) and two genetic variants with glutamine substitutions at lysine residues 114 and 139 were expressed in insect cells using a baculovirus-driven expression system. The purified proteins were used to evaluate the potential role(s) of these residues in the pentasaccharide-mediated activation of ATIII. The second order rate constants for the inhibition of factor Xa by both of the genetic variants were nearly identical to those of recombinant native ATIII, indicating that the glutamine substitutions did not result in serious protein conformational changes. The glutamine substitution at lysine 139 had no effect on the pentasaccharide-mediated activation of ATIII toward factor Xa. In contrast, lysine 114 was found to be critical in the activation of ATIII toward factor Xa. No activation was observed, even at a pentasaccharide concentration 10 times higher than that required to activate recombinant native ATIII. These data are the first to demonstrate a pivotal role for lysine 114 in the pentasaccharide-mediated activation of ATIII.

We have used purified protease nexin-I (PN-I) from human fibroblasts to develop a polyclonal antibody that specifically blocks the PN-I-mediated cellular binding of thrombin and urokinase. Anti-PN-I IgG did not inhibit the binding of 125I-epidermal growth factor-binding protein to fibroblasts, which is mediated by protease nexin-II, another cell-secreted, serine protease inhibitor that is distinct from PN-I. This furthers the belief that the protease nexins are distinct from one another. In addition, while anti-PN-I IgG immunoprecipitated PN-I X thrombin complexes, it did not do so with antithrombin-III X thrombin. Metabolically labeled PN-I was also immunoprecipitated by IgG, indicating that the protein can be labeled in vivo. The antibody also recognized primarily one band on Western transfers of conditioned medium from fibroblast cultures. These results suggest that anti-PN-I will be useful in probing the physiological role of PN-I as well as its biosynthesis.

Protease nexin-I (PN-I) is representative of a newly described class of serine protease inhibitors secreted by human fibroblasts, the protease nexins. Protease nexins form covalent complexes with their target proteases, subsequently binding to cells via specific receptors. PN-I preferentially binds thrombin, urokinase, trypsin, and plasmin, and its binding to thrombin is accelerated by heparin. We have previously described the production of a polyclonal antibody against PN-I which is able to block the binding of PN-I X proteinase complexes to cells and will immunoprecipitate metabolically labeled PN-I. Anti-PN-I was used to investigate the biosynthesis and regulation of PN-I in human fibroblasts. Unlabeled PN-I could compete for the binding of metabolically labeled PN-I to anti-PN-I, as shown by the elimination of the 43-kDa band representing PN-I on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographs. Excision of this 43-kDa band from gels, followed by amino-terminal sequencing, showed a homogeneous protein that is homologous with that described by Scott et al. (Scott, R. W., Bergman, B. L., Bajpai, A., Hersh, R. T., Rodriguez, H., Jones, B. N., Barreda, C., Watts, S., and Baker, J. B. (1985) J. Biol. Chem. 260, 7029-7034). An analysis of the biosynthesis of the PN-I revealed that a lower Mr precursor exists intracellularly. This apparent rough endoplasmic reticulum form appears as a doublet on sodium dodecyl sulfate gels, as does mature PN-I. The PN-I precursor was also sensitive to endoglycosidase H, suggesting that it contains N-linked carbohydrates of the high mannose form. Mature PN-I is not sensitive to endoglycosidase H, but does contain 3 kDa of N-linked carbohydrate. PN-I appears to be constitutively secreted by fibroblasts. PN-I levels in conditioned media reach a steady state within 48 h, although PN-I synthesis maintains a constant rate. This steady state is due to the continuous uptake of PN-I from medium, presumably through a specific receptor.

A heparin-binding peptide within antithrombin III (ATIII) was identified by digestion of ATIII with Staphylococcus aureus V8 protease followed by purification on reverse-phase high pressure liquid chromatography using a C-4 column matrix. The column fractions were assayed for their ability to bind heparin by ligand blotting with 125I-fluoresceinamine-heparin as previously described (Smith, J. W., and Knauer, D. J. (1987) Anal. Biochem. 160, 105-114). This analysis identified at least three fractions with heparin binding ability of which the peptide eluting at 25.4 min gave the strongest signal. Amino acid sequence analysis of this peptide gave a partially split sequence which was consistent with regions encompassing amino acids 89-96 and 114-156. These amino acids are present in a 1:1 molar ratio which is consistent with a disulfide linkage between Cys-95 and Cys-128. High affinity heparin competed more effectively for the binding of 125I-fluoresceinamine-heparin to this peptide than low affinity heparin. Chondroitin sulfate did not block the binding of 125I-fluoresceinamine-heparin to the peptide. These data strongly suggest that the isolated peptide represents a native heparin-binding region within intact ATIII. Computer generation of a plot of running charge density of ATIII confirms that the region encompassing amino acid residues 123-141 has the highest positive charge density within the molecule. A hydropathy plot of ATIII was generated using a method similar to that of Kyte and Doolittle (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132). This plot indicates that amino acid residues 126-140 are exposed to the exterior surface of the molecule. Based on these data, we suggest that the region corresponding to amino acid residues 114-156 is a likely site for the physiological heparin-binding domain of ATIII. We also conclude that the proposed disulfide bridges within the protein are suspect and should be re-examined (Petersen, T. E., Dudek-Wojiechowska, G., Sottrup-Jensen, L., and Magnussun, S. (1979) in The Physiological Inhibitors of Coagulation and Fibrinolysis (Collen, D., Wiman, B., and Verstaeta, M., eds) pp. 43-54, Elsevier Scientific Publishing Co., Amsterdam).

Epidermal growth factor carrier protein (CP) is an arginine endopeptidase bound to epidermal growth factor (EGF) in vivo that processes pro-EGF to EGF and potentiates EGF action. Here, we provide a base for studying the biological functions of CP by showing that highly purified 125I-labeled CP, free of contaminating EGF, is specifically bound and internalized by normal human fibroblasts in serum-free medium. The characteristics of the binding reaction, however, were unusual and not consistent with direct interaction of CP with cell surface receptors. Subsequent experiments showed that cellular binding of 125I-labeled CP was mediated via a cell-secreted protein. We named the protein carrier protein nexin (CPN) because of its close functional similarity to protease nexin, which mediates cellular binding of thrombin or urokinase. Both CPN and protease nexin are secreted by cells, form covalent complexes with regulatory proteases in the extracellular environment, and mediate cellular binding of these proteases, apparently via a cell surface receptor for the nexin moiety of the complex. By several criteria, however, CPN and protease nexin are unique entities. This finding of a specific interaction of a growth factor carrier protein with cells suggests the possibility of additional physiological functions for these carriers in growth factor action or metabolism or both.

Because mitogenic stimulation by epidermal growth factor (EGF) requires at least an 8--12-h exposure to commit the majority of the cells to divide, a number of complicating processes must be evaluated in an analysis of the relationship between receptor occupancy and the biological response. During this period, the number of cellular EGF receptors changes and the cells continuously bind, internalize, and degrade the hormone. To deal with these changes, we utilized our previously described steady state model that permits an analysis of EGF interactions with cells under steady state conditions. For these studies we derived and measured a number of steady state rate constants to describe the interaction of 125I-EGF with cultured human and mouse fibroblast-like cells. The validity of these constants was established by demonstrating the close agreement between experimentally determined results and predicted results from computer simulations using these constants. These approaches in conjunction with experiments on the mitogenic stimulation of the cells with EGF led to the conclusion that there is a linear relationship between mitogenic stimulation and EGF receptor occupancy at steady state, with no apparent "spare" EGF receptors.