Sensory and motor neuron-derived factor is a transmembrane heregulin that is expressed on the plasma membrane with the active domain exposed to the extracellular environment.

The beta-heregulin sensory and motor neuron-derived factor (SMDF) has been suggested to be an important regulator of Schwann cell development and proliferation. In the present study, human SMDF was expressed in cultured cell lines. The cells and the recombinant protein were used to examine the membrane association and biological activity of the growth factor. Transfection of cells with SMDF cDNA constructs bearing FLAG epitope tags at either the amino- or carboxyl-terminal ends of the polypeptide resulted in expression of anti-FLAG immunoreactive polypeptides of approximately 44 and 83 kDa. The 83-kDa polypeptide was the major form expressed on the cell surface, as demonstrated by sensitivity to proteolysis in intact cells and surface biotinylation. SMDF was tightly associated with membranes isolated from transfected cells but was solubilized by Triton X-100. Immunofluorescent staining and immunoprecipitation experiments using cells expressing amino- or carboxyl-terminal tagged SMDF revealed that only the carboxyl-terminal end of the protein is exposed on the cell surface. Membranes from SMDF-transfected cells stimulated tyrosine phosphorylation of the beta-heregulin receptor ErbB3 in Schwann cells. Conditioned medium from transfected cells contained a similar activity, suggesting that SMDF is subject to proteolytic release from the plasma membrane. In contrast with other beta-heregulin isoforms, SMDF failed to bind heparin. Stimulation of Schwann cell ErbB3 receptor phosphorylation by SMDF was not affected by inhibition of Schwann cell heparan sulfate proteoglycan synthesis. These results demonstrate that SMDF is a type II transmembrane protein. This orientation places the active epidermal growth factor homology domain, which is located near the carboxyl-terminal end of the polypeptide, on the cell surface, where it can function as a membrane-anchored growth factor.

Synergistic regulation of Schwann cell proliferation by heregulin and forskolin.

A peptide corresponding to the epidermal growth factor homology domain of beta-heregulin stimulated autophosphorylation of the heregulin receptors erbB2 and erbB3 in Schwann cells and activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2. Heregulin-dependent activation of PAK65, a component of the stress-activated signaling pathway, ribosomal S6 kinase, and a cyclic AMP (cAMP) response element binding protein (CREB) kinase, identified as p95(RSK2), was also observed. Receptor phosphorylation and activation of these kinases in response to heregulin occurred in the absence of forskolin stimulation and were not augmented in cells treated with forskolin, a direct activator of adenylyl cyclase. Schwann cell proliferation in response to heregulin was observed only when the cells were also exposed to an agent that elevates cAMP levels. In the absence of heregulin, elevation of cAMP levels failed to stimulate Schwann cell proliferation. Forskolin significantly enhanced heregulin-stimulated expression of cyclin D and phosphorylation of the retinoblastoma gene product. In cells treated with both heregulin and forskolin there was a sustained accumulation of phospho-CREB, which was not observed in cells treated with either agent alone. Heregulin and forskolin synergistically activated transcription of a cyclin D promoter construct. These results demonstrate that heregulin-stimulated activation of MAP kinase is not sufficient to induce maximal Schwann cell proliferation. Expression of critical cell cycle regulatory proteins and cell division require activation of both heregulin and cAMP-dependent processes.

Subunit expression of signal transducing G proteins in cardiac tissue: implications for phospholipase C-beta regulation.

In the heart, alpha-adrenergic, angiotensin II and endothelin signaling pathways modulate short-term changes in chronotropy and inotropy, and participate in the long-term control of cardiac growth. A shared feature of these signaling pathways is the stimulation of phosphatidylinositol (PI) turnover, which is thought to occur via G protein-mediated regulation of phospholipase C (PLC) activity. However, G protein subunits capable of regulating PLC activity have not been identified in different regions and cell types of the heart and members of the G protein-regulated PLC-beta isozyme family have not been documented in the heart. Using a battery of antipeptide specific antisera directed against the G protein alpha q, beta and gamma subunit families and against members of the PLC-beta, PLC-gamma and PLC-delta families, we demonstrated that heart tissues express the G protein alpha subunits alpha q and alpha 11, multiple G protein beta and gamma subunits, and PLC-beta 3, a phospholipase C isozyme regulated by either G protein alpha or beta gamma subunits. The degree of expression and distribution of these subunits differed between regions of the heart (atria versus ventricle) and changed with development. These data lay the ground work for future studies to determine the functional coupling of specific subsets of these components involved in receptor activation of PI turnover in the heart.

Localization of a heterotrimeric G protein gamma subunit to focal adhesions and associated stress fibers.

Signal transducing heterotrimeric G proteins are responsible for coupling a large number of cell surface receptors to the appropriate effector(s). Of the three subunits, 16 alpha, 4 beta, and 5 gamma subunits have been characterized, indicating a potential for over 300 unique combinations of heterotrimeric G proteins. To begin deciphering the unique G protein combinations that couple specific receptors with effectors, we examined the subcellular localization of the gamma subunits. Using anti-peptide antibodies specific for each of the known gamma subunits, neonatal cardiac fibroblasts were screened by standard immunocytochemistry. The anti-gamma 5 subunit antibody yielded a highly distinctive pattern of intensely fluorescent regions near the periphery of the cell that tended to protrude into the cell in a fibrous pattern. Dual staining with anti-vinculin antibody showed co-localization of the gamma 5 subunit with vinculin. In addition, the gamma 5 subunit staining extended a short distance out from the vinculin pattern along the protruding stress fiber, as revealed by double staining with phalloidin. These data indicated that the gamma 5 subunit was localized to areas of focal adhesion. Dual staining of rat aortic smooth muscle cells and Schwann cells also indicated co-localization of the gamma 5 subunit and vinculin, suggesting that the association of the gamma 5 subunit with areas of focal adhesion was wide-spread.

Metal ion binding to dog osteocalcin studied by 1H NMR spectroscopy.

One-dimensional 1H NMR was employed to study the effects of Ca2+ and Lu3+ binding on the apo and calcium-saturated forms of dog bone Gla protein (BGP, osteocalcin). Titration of apo dog BGP with Ca2+ in 20 mM NaCl showed spectral perturbations consistent with the binding of 5 mol equiv of calcium in the NMR slow-exchange limit. The first 2 Ca2+ equiv induced significant conformational changes in the apoprotein, binding cooperatively with a Kd1 approximately 5.0 x 10(-4) M and a Hill coefficient H = 2.3 in 20 mM NaCl. The last 3 equiv bound with a slightly weaker affinity and did not induce significant structural changes. Neither the affinity nor the stoichiometry of calcium binding was significantly altered at 150 mM NaCl. The addition of only 1 Lu3+ equiv to apo dog osteocalcin was sufficient to induce the same spectral perturbations as 2 Ca2+ ions. The addition of 2 Lu3+ equiv to calcium-saturated osteocalcin had little effect on its 1H NMR spectrum, and BGP aggregated at [Lu3+]o/[BGP]o ratios greater than 2 in either the presence or absence of calcium. The spectrum of calcium-saturated osteocalcin was invariant at < or = 55 degrees C (< or = 50 degrees C in 150 mM NaCl), after which the proton resonances shifted to frequencies more characteristic of apo BGP. Saturation with calcium somewhat stabilized the apo dog osteocalcin protein against conformational changes induced at pH extremes; apo BGP was stable at 6.0 < or = pH < or = 10, and calcium-saturated BGP was stable at 5.8 < or = pH < or = 10. Both our NMR and gel filtration data indicate that calcium-saturated osteocalcin exists as a dimer at both high and low protein concentrations. A conformational change in dog osteocalcin was thus induced by the cooperative association of Ca2+ to two high-affinity sites on the protein and stabilized by the association of 3 additional Ca2+ equiv. The results of our temperature and calcium binding studies were consistent with an estimated Kd1 approximately 5.0 x 10(-4) M for the two high-affinity sites. Lutetium induced the same structural changes in osteocalcin as calcium, but the two high-affinity Ca2+ binding sites did not have equal affinities for Lu3+. The BGP:Ca2+ complex was unstable at the low pH conditions induced by osteoclasts during bone resorption, yet the osteocalcin protein retained a BGP:Ca(2+)-like conformation at low pH. However, unlike the calcium-saturated form of the protein, osteocalcin was monomeric at low pH.