Dana C. Mahadeo

Nerve growth factor (NGF) binds to two cell surface receptors, p140trk and p75NGFR, which are both expressed in responsive sensory, sympathetic, and basal forebrain cholinergic neurons. While p140trk belongs to the family of receptor tyrosine kinases, p75NGFR is a member of the TNF/Fas/CD40/CD30 family of receptors. Current views of neurotrophin receptor function have tended to interpret p140trk as the high affinity NGF-binding site. To assess if the binding of NGF to p140trk was distinguishable from binding to high affinity sites on neuronal cells, PC12 cell sublines were generated which expressed p140trk alone, or coexpressed both p140trk and p75NGFR. Kinetic analysis of 125I-NGF binding indicates that it has an unusually slow rate of association with p140trk (k + 1 = 8 x 10(5) M-1 s-1). When both p140trk and p75NGFR receptors are coexpressed, the rate of association of NGF is increased 25-fold to produce a higher affinity binding site. An increase in the rate of internalization was also observed. Since high affinity binding and internalization are believed to be prerequisite for the biological activities of NGF, these results suggest that the biological effects by NGF are derived from a novel kinetic binding site that requires the expression of both receptors. The implications of these results with respect to multisubunit polypeptide receptors are discussed.

The neurotrophins, a family of related polypeptide growth factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-3 and NT-4/5 promote the survival and differentiation of distinctive sets of embryonic neurons. Here we define a new functional role for neurotrophins, as autocrine or local paracrine mediators of vascular smooth muscle cell migration. We have identified neurotrophins, and their cognate receptors, the trk tyrosine kinases, in human and rat vascular smooth muscle cells in vivo. In vitro, cultured human smooth muscle cells express BDNF; NT-3; and trk A, B, and C. Similarly, rat smooth muscle cells expressed all three trk receptors as well as all four neurotrophins. Moreover, NGF induces cultured human smooth muscle cell migration at subnanomolar concentrations. In the rat aortic balloon deendothelialization model of vascular injury, the expression of NGF, BNDF, and their receptors trk A and trk B increased dramatically in the area of injury within 3 days and persisted during the formation of the neointima. In human coronary atherosclerotic lesions, BDNF, NT-3, and NT-4/5, and the trk B and trk C receptors could be demonstrated in smooth muscle cells. These findings suggest that neurotrophins play an important role in regulating the response of vascular smooth muscle cells to injury.

The transforming gene of the avian sarcoma virus CT10 encodes a fusion protein (p47gag-crk or v-Crk) containing viral Gag sequences fused to cellular sequences consisting primarily of Src homology regions 2 and 3 (SH2 and SH3 sequences). Here we report a novel function of v-Crk in the mammalian pheochromocytoma cell line, PC12, whereby stable expression of v-Crk induces accelerated differentiation, as assessed by induction of neurites following nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) treatment compared with the effect in native PC12 cells. Surprisingly, however, these cells also develop extensive neurite processes after epidermal growth factor (EGF) stimulation, an event which is not observed in native PC12 cells. Following EGF or NGF stimulation of the v-CrkPC12 cells, the v-Crk protein itself became tyrosine phosphorylated within 1 min. Moreover, in A431 cells or TrkA-PC12 cells, which overexpress EGF receptors and TrkA, respectively, a GST-CrkSH2 fusion protein was indeed capable of binding these receptors in a phosphotyrosine-dependent manner, suggesting that v-Crk can directly couple to receptor tyrosine kinase pathways in PC12 cells. In transformed fibroblasts, v-Crk binds to specific tyrosine-phosphorylated proteins of p130 and paxillin. Both of these proteins are also complexed to v-Crk in PC12 cells, as evidenced by their coprecipitation with v-Crk in detergent lysates, suggesting that common effector pathways may occur in both cell types. However, whereas PC12 cellular differentiation can occur solely by overexpression of the v-Src or oncogenic Ras proteins, that induced by v-Crk requires a growth factor stimulatory signal, possibility in a two-step process.

Upon removal of nutrients, the amoebae of the cellular slime mold Dictyostelium discoideum differentiate into dormant spores which survive starvation stress. In this study, we demonstrate that half of the actin molecules in the spores are tyrosine-phosphorylated. The phosphorylated actin is distributed around immobile crenate mitochondria and vesicles, as well as in the cytoplasm of the spores. The actin isolated from spore lysates contains phosphorylated and unphosphorylated forms at the same molar ratio as that of the original whole spore lysate. Under actin polymerizing conditions they form actin filaments and then they are completely depolymerized under actin depolymerizing conditions, indicating that tyrosine phosphorylation of actin may not prohibit actin polymerization nor stimulate depolymerization. The phosphorylation levels increase at the end of the culmination stage when spores have matured morphologically and physiologically, and reach maximum levels after an additional 12 hours of development. The levels are stable for 20 days following spore maturation, and decline to undetectable levels within the next 10 days. Spores having high levels of phosphorylation show high viability, and vice versa. Following activation of spores with nutrient medium containing spore germination promoters, the phosphorylation levels quickly decrease with a half-life of about 5 minutes. After 20 minutes spores begin to swell. At this later time, most of the phosphorylated actin already has been dephosphorylated. Also, in heat-activated spores actin dephosphorylation occurs prior to spore swelling. However, addition of phosphatase inhibitors following heat-activation, prevented spore swelling and dephosphorylation of actin. Our data indicate that the high levels of actin tyrosine phosphorylation, specific to the spore stage, may be required for maintaining dormancy to withstand starvation stress. The rapid dephosphorylation of actin leads to a reactivated dynamic actin system which participates in spore swelling, vesicle movement, and mitochondrial shape changes during the spore germination process.