Down-regulation of a 67-kDa YIGSR-binding protein upon differentiation of human neuroblastoma cells.

Differentiated human neuroblastoma LA-N1 cells that were exposed to dibutyryl adenosine 3',5'-cyclic monophosphate for 5 days (primed cells) showed increased adhesion to laminin-, fibronectin-, and collagen type I-coated plates as compared to unprimed cells. Moreover, primed cells seemed to adhere best to laminin. The binding site in laminin, mediating cell attachment, was identified as containing the YIGSR sequence, a known cell binding motif, located in the short arm of the B1 chain of laminin. The synthetic peptide amide, C(YIGSR)3-NH2, containing a repeat of this binding motif, inhibited the attachment of neuroblastoma cells to laminin in a competitive manner, and its inhibitory activity was inversely dependent on laminin concentrations. Affinity chromatography of membrane-extracted proteins over an Affi-Gel 10 column conjugated to C(YIGSR)3-NH2, revealed a major YIGSR-binding protein with an apparent molecular mass of 67 kDa. The 67-kDa surface membrane protein was specifically eluted from the column with the soluble C(YIGSR)3-NH2 peptide, but not with an unrelated peptide. Furthermore, no 67-kDa laminin-binding protein was recovered from an unrelated peptide matrix with the free C(YIGSR)3-NH2 peptide. Ligand blot overlay assays with biotin-labeled C(YIGSR)3-NH2 peptide demonstrated that the 67-kDa receptor is indeed a YIGSR-binding protein. This 67-kDa laminin-binding protein appeared to be down-regulated upon differentiation of LA-N1 cells, as indicated by the level of this protein and its mRNA.

Signaling mechanisms controlled by melanocortins in melanoma, lacrimal, and brain astroglial cells.

Melanocortins appear to be involved as regulators in an ever growing number of physiological processes in cells and tissues of diverse functions. While such trends are apparent also in the case of other peptide hormones, it appears that melanocortin receptors can be regarded as unique among G-protein-linked receptors due to their special need for extracellular Ca2+ which may relate to some, yet undetermined selectivity of their actions. The physiological role that Ca2+ may be playing and the diverse signaling mechanisms regulated, as well as the nature of the cell-specific responses elicited in melanocortin-sensitive cells/tissues, have yet to be elucidated. Likewise, it will be of interest to establish the relationship of melanocortins to processes like growth and differentiation of cells, as well as to higher, more complex processes such as those regulated in the CNS.

Phosphorylation state of the GLUT4 isoform of the glucose transporter in subfractions of the rat adipose cell: effects of insulin, adenosine, and isoproterenol.

The acute effects of insulin, adenosine, and isoproterenol on the activity, subcellular distribution, and phosphorylation state of the GLUT4 glucose transporter isoform were investigated in rat adipocytes under conditions carefully controlled to monitor changes in cAMP-dependent protein kinase (A-kinase) activity. In contrast to GLUT1, which has not been shown to be phosphorylated even when cells are exposed to any of the above agents, GLUT4 was partially phosphorylated (0.1-0.2 mol/mol) when the activity of the A-kinase was suppressed, and remained unchanged in response to insulin. Isoproterenol elicited a 64% inhibition of insulin-stimulated glucose transport activity in the absence, but not the presence, of adenosine receptor agonists. However, in either the presence or the absence of agonists, A-kinase was activated as assessed by examining the phosphorylation of the major adipocyte A-kinase substrate, perilipin. Similarly, under either condition, phosphorylation of GLUT4 was enhanced 1.4-fold in the intracellular membranes, but no significant change was observed in the plasma membrane. In the absence of adenosine receptor agonists, isoproterenol exerted a small (14%) but significant inhibition of the insulin-induced translocation of GLUT4 but had no effect on the translocation of GLUT1. Thus, changes in the phosphorylation state and/or subcellular distribution of GLUT4 cannot account for the inhibition of insulin-stimulated glucose activity induced by isoproterenol.

Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets.

The lipid fraction ("fat cake") of rat epididymal adipocytes contains a prominent phosphoprotein (62 kDaapp by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) that is multiply phosphorylated by cAMP-dependent protein kinase in vivo, at which point it migrates as a 65/67-kDaapp doublet by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is by far the most heavily radiolabeled protein in the cell. Western blot analysis of various tissues with immunopurified antibodies purified from antisera raised against the 62-kDa species suggests that the protein is specific for adipocytes. This protein, which we term perilipin, is found in differentiated cultured 3T3-L1 adipocytes, but not in their precursor 3T3-L1 fibroblasts. Immunocytochemical studies with specific antiserum shows that the perilipin is closely associated with the periphery of lipid storage droplets in cultured adipocytes. Given its adipocyte specificity, acute regulation by hormones, and subcellular location, we speculate that perilipin plays a role in the specialized lipid storage function of adipocytes.

The melanocortin receptor in the rat lacrimal gland: a model system for the study of MSH (melanocyte stimulating hormone) as a potential neurotransmitter.

The melanocortin receptors in intraorbital and extraorbital rat lacrimal glands were studied with [125I][Nle4,D-Phe7]alpha MSH as radioligand and with several unlabeled melanocortin peptides. The pharmacological properties of the melanocortin receptor in both tissues appeared to be essentially identical. Receptor binding was studied in a membrane fraction sedimented at 12,000-100,000 X g, establishing for [125I][Nle4,D-Phe7]alpha MSH a KD of 0.76 and 2.2 nM for the intra- and extraorbital glands, respectively. Binding of the radioligand was competitively inhibited by alpha MSH (alpha-melanocyte stimulating hormone) and ACTH-(1-24) with IC50 values in the submicromolar range. MSH binding in both tissues was abolished by EGTA and was increased dose dependently with elevation of free Ca2+ ion concentration. The half-maximal effect on MSH binding was obtained around 200 microM Ca2+ and maximal binding was reached at nearly 2 mM free Ca2+ in membrane preparations from both tissues. The calmodulin-binding peptides, melittin, mastoparan and M5, the latter being the 18-amino acid synthetic analogue of the C-terminal calmodulin-binding domain of myosin light chain kinase, inhibited MSH binding in the concentration range of 1-20 microM. Macroscopic autoradiographic analysis of cryosections prepared from either lacrimal gland to which [125I][Nle4,D-Phe7]alpha MSH was subsequently bound, showed the melanocortin receptor to be uniformly distributed within the acinar lobes. At the microscopic level, MSH was found to be associated with the acinar cells, primarily at the basal perinuclear region. Peroxidase secretion from extraorbital lacrimal slices was stimulated by MSH, epinephrine and carbamylcholine to a similar extent. The response of the tissue to stimulation by MSH was however not blocked by alpha/beta-adrenoceptor blockers or by atropine, suggesting that MSH acts as a primary secretagogue in this tissue. Thus, this system seems to be uniquely suited to serve as a model for the study of both the molecular and pharmacological details of the action of MSH and other melanocortins in a non-melanogenic tissue.

G-proteins in mammalian gametes: an immunocytochemical study.

The presence of transductory GTP(G)-regulatory proteins in mammalian gametes has been examined by indirect fluorescence immunocytochemistry. Using rabbit antisera to bovine rod beta gamma-transducin (RA beta gamma T), bovine rod holotransducin (AS-1), bovine rod alpha-transducin (RA alpha T), synthetic bovine rod alpha-transducin C-terminal decapeptide (AS-6), bovine brain alpha 39Go (RA alpha 39), and two mouse monoclonal antibodies raised against frog retinal transducin (4A), and rat brain beta-tubulin, we demonstrated the presence of corresponding immunoreactive material in both rat oocytes and bovine ejaculated sperm. Immunostaining in the oocyte was evenly distributed on the oolemma, excluding the cell cytoplasm and zona pellucida. Immunoreactive material was also present in the cumulus cells that encapsulate the oocyte. In contrast, the immunofluorescence corresponding to transductory G-proteins was confined in sperm to functionally defined regions in the head and tail, in a manner specific for each antibody. While RA beta gamma T, AS-1 and RA alpha 39 all stained the entire acrosome, AS-6 and RA alpha T stained only the acrosomal tip. Monoclonal antibody 4A stained the midpiece exclusively and anti-rat betaq-tubulin (a structural G-protein) stained the full length of the sperm tail. The existence of several G-protein types in mammalian gametes suggests their possible involvement in the regulation of various effector systems, in a manner reminiscent of somatic cells. The unique situation in sperm, where different G-proteins show distinct and specific patterns of distribution, further suggests their association with various effector systems in discrete functional domains.

Stimulation of partially purified adenylate cyclase from bull sperm by bicarbonate.

Solubilized and partially purified adenylate cyclase from bull sperm was found to be specifically activated (up to 6-fold) by sodium bicarbonate (NaHCO3) and to a lesser extent by NaNO3. Other sodium salts were either ineffective (e.g. NaCOOH) or inhibitory (e.g. NaHSO3, NaHSO4 and Na2B4O7). Stimulation by NaHCO3 was dose-dependent in the range of 0-40 mM and was greater when enzyme activity was assayed in the presence of magnesium as compared with manganese ions. Bicarbonate seems to affect maximal enzyme velocity (Vmax) and has no effect on the Km of adenylate cyclase for Mn-ATP. Stimulation of adenylate cyclase by NaHCO3 coincided with the elution pattern of the enzyme as recorded following chromatography on DEAE-cellulose or gel filtration on BioGel P-100. These results suggest that in the course of stimulation of sperm adenylate cyclase, bicarbonate is likely to interact directly with the enzyme. Furthermore, this intrinsic and unique property of sperm adenylate cyclase may explain results reported by others on the stimulation of cAMP production by bicarbonate in intact and broken sperm preparations and suggest a biochemical basis for enhanced sperm motility associated with high bicarbonate concentrations.

Donnan potentials from striated muscle liquid crystals. Lattice spacing dependence.

Electrochemical potentials were measured as a function of myofilament packing density in crayfish striated muscle. The A-band striations are supramolecular smectic B1 lattice assemblies of myosin filaments and the I-band striations are nematic liquid crystals of actin filaments. Both A- and I-bands generate potentials derived from the fixed charge that is associated with structural proteins. In the reported experiments, filament packing density was varied by osmotically reducing lattice volume. The electrochemical potentials were measured from the A- and I-bands in the relaxed condition over a range of lattice volumes. From the measurements of relative cross-sectional area, unit-cell volume (obtained by low-angle x-ray diffraction) and previously determined effective linear charge densities (Aldoroty, R.A., N.B. Garty, and E.W. April, 1985, Biophys. J., 47:89-96), Donnan potentials can be predicted for any amount of compression. In the relaxed condition, the predicted Donnan potentials correspond to the measured electrochemical potentials. In the rigor condition, however, a net increase in negative charge associated with the myosin filament is observed. The predictability of the data demonstrates the applicability of Donnan equilibrium theory to the measurement of electrochemical potentials from liquid-crystalline systems. Moreover, the relationship between filament spacing and the Donnan potential is consistent with the concept that surface charge provides the necessary electrostatic force to stabilize the myofilament lattice.

Donnan potentials from striated muscle liquid crystals. Sarcomere length dependence.

Donnan potentials from A-bands and I-bands were measured as a function of sarcomere length in skinned long-tonic muscle fibers of the crayfish. These measurements were made using standard electrophysiological technique. Simultaneously, the relative cross-sectional area of the fibers was determined. Lattice plane spacings and hence unit-cell volumes were determined by low-angle x-ray diffraction. At a sarcomere length at which the myosin filaments and actin filaments nominally do not overlap, measurements of potential, relative cross-sectional area, and unit-cell volume were used in conjunction with Donnan equilibrium theory to calculate the effective linear charge densities along the myosin filament (6.6 X 10(4) e-/mu) and actin filament (6.8 X 10(3) e-/mu). Using these linear charge densities, unit-cell volumes and Donnan equilibrium theory, an algorithm was developed to predict A-band and I-band potentials at any sarcomere length. Over the range of sarcomere lengths investigated, the predicted values coincide with the experimental data. The ability of the model to predict the data demonstrates the applicability of Donnan equilibrium theory to measurements of electrochemical potential from liquid-crystalline systems.