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The very low density lipoprotein receptor (VLDLR) binds, among other ligands, the Mr 40,000 receptor-associated protein (RAP) and a variety of serine proteinase-serpin complexes, including complexes of the proteinase urokinase-type plasminogen activator (uPA) with the serpins plasminogen activator inhibitor-1 (PAI-1) and protease nexin-1 (PN-1). We have analyzed the binding of RAP, uPA.PAI-1, and uPA.PN-1 to two naturally occurring VLDLR variants, VLDLR-I, containing all eight complement-type repeats, and VLDLR-III, lacking the third complement-type repeat, encoded by exon 4. VLDLR-III displayed approximately 4-fold lower binding of RAP than VLDLR-I and approximately 10-fold lower binding of the most C-terminal one of the three domains of RAP. In contrast, the binding of uPA.PAI-1 and uPA.PN-1 to the two VLDLR variants was indistinguishable. Surprisingly, uPA.PN-1, but not uPA.PAI-1, competed RAP binding to both VLDLR variants. These observations show that the third complement-type repeat plays a crucial role in maintaining the contact sites needed for optimal recognition of RAP, but does not affect the proteinase-serpin complex contact sites, and that two ligands can show full cross-competition without sharing the same contacts with the receptor. These results elucidate the mechanisms of molecular recognition of ligands by receptors of the low density lipoprotein receptor family.

Brain cells and glioma cells in culture release a protein which induces neurite outgrowth in neuroblastoma cells. This neurite‐promoting factor (NPF), which has been purified from serum‐free glioma conditioned medium, has an apparent mol. wt. of 43 000. NPF inhibits urokinase as well as plasminogen activator‐dependent caseinolysis or fibrinolysis. NPF and urokinase form an SDS‐resistant complex. The fact that this glia‐derived NPF is a potent protease inhibitor indicates that glial cells modulate the proteolytic activity associated with neuronal cells and suggests that this phenomenon is one of the biochemical events involved in the regulation of neurite growth.

Cultured explants from early postnatal mouse cerebellum were used to examine the influence of a 43-kDa glia-derived neurite-promoting factor (GdNPF) on the migration of [3H]thymidine-labeled granule cell neurons. GdNPF, which is a potent serine protease inhibitor, significantly reduced the extent of granule cell migration in a dose-dependent manner. This effect could be neutralized by addition of thrombin, which binds GdNPF. Other protease inhibitors such as aprotinin, hirudin, soybean trypsin inhibitor, leupeptin, 6-aminocaproic acid, and D-Phe-Pro-ArgCH2Cl do not show this inhibitory effect. These results demonstrate that a glia-derived protein can regulate the migration of postmitotic neurons, an important cellular event in the development of the nervous system.

Cultured myoblasts derived from limb muscle of newborn rats express thrombin receptor immunoreactivity on their surface. Receptor expression is repressed upon myoblast fusion. This is due at least in part to a decrease in the amount of the thrombin receptor mRNA. Addition of thrombin triggers calcium transients only in mono- but not multinucleated muscle cells. Furthermore, thrombin increases the rate of myoblast proliferation that coincides with an activation of mitogen-activated protein kinase. Northern analysis of thrombin receptor mRNA expression in skeletal muscle showed that the transcript is present at a relatively high level at birth, but is almost undetectable in the adult. By in situ hybridization, the mRNA at birth appeared to be present mostly in mononucleated cells grouped in clusters, but not in muscle fibers. Very few nuclei surrounded by a mRNA signal were present on muscle sections of rats 24 days postnatally. These results suggest that the thrombin receptor plays a role in muscle development.

Neuroblastoma cells have been found to extend neurites when grown in a medium supplemented with delipidated fetal calf serum. Fetal calf sera from different commercial sources give rise to marked differences in the degree of this spontaneous morphological differentiation. The phenomenon can be prevented by the addition of certain fatty acids; oleic acid is especially effective. The serum-free conditioned medium from glial cells can quantitatively antagonize the effect of oleic acid, suggesting that glial factor activity could be due to components [lipids and/or macromolecular factors(s)] that are able to modify the properties of the neuroblastoma cell membrane.

Site-directed mutagenesis was used to investigate the role of basic residues in the thrombin anion-binding exosite-I during formation of thrombin-antithrombin III (ATIII), thrombin-protease nexin 1 (PN1), and thrombin-heparin cofactor II (HCII) inhibitor complexes, in the absence and presence of glycosaminoglycans. In the absence of glycosaminoglycan, association rate constant (kon) values for the inhibition of the mutant thrombins (R35Q, K36Q, R67Q, R73Q, R75Q, R77(a)Q, K81Q, K109Q, K110Q, and K149(e)Q) by ATIII and PN1 were similar to wild-type recombinant thrombin (rIIa), whereas kon values were decreased 2-3-fold for HCII against the majority of the exosite-I mutants. The exosite-I mutants did not have a significant effect on heparin-accelerated inhibition by ATIII with maximal kon values similar to rIIa. A small effect was seen for PN1/heparin inhibition of the exosite-I mutants R35Q, R67Q, R73Q, R75Q, and R77(a)Q, where kon values were decreased 2-4-fold, compared with rIIa. For HCII/heparin, kon values for inhibition of the exosite-I mutants (except R67Q, R73Q, and K149(e)Q) were 2-3-fold lower than rIIa. Larger decreases in kon values for HCII/heparin were found for R67Q and R73Q thrombins with 441- and 14-fold decreases, respectively, whereas K149(e)Q was unchanged. For HCII/dermatan sulfate, R67Q and R73Q had kon values reduced 720- and 48-fold, respectively, whereas the remaining mutants were decreased 3-7-fold relative to rIIa. The results suggest that ATIII has no major interaction with exosite-I of thrombin with or without heparin. PN1 bound to heparin uses exosite-I to some extent, possibly by utilizing the positive electrostatic field of exosite-I to enhance orientation and thrombin complex formation. The larger effects of the thrombin exosite-I mutants for HCII inhibition with heparin and dermatan sulfate indicate its need for exosite-I, presumably through contact of the "hirudin-like" domain of HCII with exosite-I of thrombin.

The cyclic 3′,5′-adenosine monophosphate (c-AMP) phosphodiesterase from Escherichia colihas been partially purified. The enzyme has an apparent molecular weight of 30,000, a Michaelis constant of 0.5 mM c-AMP, and a pH optimum of 7. The partially purified enzyme requires for activity the presence of a reducing compound and of either iron or a protein which seemingly acts as iron carrier.