Functional interactions of the immunoglobulin superfamily member F11 are differentially regulated by the extracellular matrix proteins tenascin-R and tenascin-C.

The axon-associated protein F11 is a GPI-anchored member of the immunoglobulin superfamily that promotes axon outgrowth and that shows a complex binding pattern toward multiple cell surface and extracellular matrix proteins including tenascin-R and tenascin-C. In this study, we demonstrate that tenascin-R and tenascin-C differentially modulate cell adhesion and neurite outgrowth of tectal cells on F11. While soluble tenascin-R increases the number of attached cells and the percentage of cells with neurites on immobilized F11, tenascin-C stimulates cell attachment to a similar extent but decreases neurite outgrowth. The cellular receptor interacting with F11 has been previously identified as NrCAM; however, in the presence of tenascin-R or tenascin-C cell attachment and neurite extension are independent of NrCAM. Antibody perturbation experiments indicate that beta(1) integrins instead of NrCAM function as receptor for neurite outgrowth of tectal cells on an F11.TN-R complex. Cellular binding assays support the possibility that the interaction of F11 to NrCAM is blocked in the presence of tenascin-R and tenascin-C. Furthermore, a sandwich binding assay demonstrates that tenascin-R and tenascin-C are able to form larger molecular complexes and to link F11 polypeptides by forming a molecular bridge. These results suggest that the molecular interactions of F11 might be regulated by the presence of tenascin-R and tenascin-C.

Thrombin, phorbol ester, and cAMP regulate thrombin receptor protein and mRNA expression by different pathways.

Human mesangial cells have been used to study the regulation of thrombin receptor protein and mRNA expression during cross-talk between different signal transduction pathways. Persistent activation of thrombin receptor by thrombin led to homologous down-regulation of thrombin receptor protein. However, thrombin receptor mRNA expression was not affected, suggesting that increased receptor degradation is responsible for homologous down-regulation. Chronic activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and of adenylylcyclase by prostaglandin E1 (PGE1) resulted in heterologous down-regulation of thrombin receptor protein. In contrast to thrombin, PMA and PGE1 reduced in parallel thrombin receptor mRNA levels to 51% and 24% of control, respectively, indicating that heterologous down-regulation of thrombin receptor protein is, at least in part, due to inhibition of receptor mRNA expression. The mechanisms of heterologous down-regulation of thrombin receptor protein have been studied in detail and compared to homologous down-regulation. PMA-induced down-regulation was completely blocked by GF 109 203 X, an inhibitor of protein kinase C. However, the loss of thrombin receptor induced by thrombin was not prevented by GF 109 203 X, indicating that homologous regulation is not dependent on protein kinase C activation. The heterologous effect of PGE1 was mimicked by 8-bromo-cAMP, isobutylmethylxanthine, and forskolin, suggesting that an increase in intracellular cAMP level is involved in heterologous regulation. Interestingly, heterologous down-regulation induced by PGE1 seems not to require previous internalization of thrombin receptor. These data indicate that thrombin receptor protein and mRNA expression can be regulated in homologous and heterologous ways by different mechanisms.

Long-term effects of thrombin require sustained activation of the functional thrombin receptor.

Thrombin is a potent activator of human glomerular epithelial cells (HGEC). Here we compare short-term and long-term effects of thrombin and thrombin receptor agonist peptide (TRAP) which selectively activates the functional thrombin receptor. TRAP, as thrombin, increases intracellular free Ca2+ concentration and acts synergistically with growth factors possessing tyrosine kinase receptors on DNA synthesis. Thrombin induces synthesis of proteins of the fibrinolytic system and cell proliferation if it is present for at least 8 h. TRAP alone does not stimulate protein synthesis and is not mitogenic. However, in the presence of the aminopeptidase inhibitor amastatin all long-term effects of thrombin can be fully mimicked by TRAP. In conclusion, different effects of thrombin and TRAP may be related to the degradation of TRAP by cellular ectoenzymes. The recently cloned thrombin receptor accounts for early intracellular signals and long-term cellular effects that require sustained activation of this receptor.