Reversible palmitoylation of signaling proteins.

Palmitoylation is unique among lipid modifications of proteins in that it is reversible and regulable. Recent advances in the study of palmitoylation include the following: the correlation of this modification with the localization of a signaling protein to specific membrane subdomains; the demonstration of a specific protein-protein interaction that is promoted by palmitoylation; and the identification, characterization, and purification of enzymes catalyzing this modification.

Interactions of thrombospondin with extracellular matrix proteins: selective binding to type V collagen.

Thrombospondin (TS), a protein first described in platelets, was recently shown to be synthesized and secreted by endothelial cells, fibroblasts, and smooth muscle cells. The presence of TS in the extracellular matrix of cultured cells has prompted us to examine the associations of this protein with matrix macromolecules. Interactions of TS with both matrix and serum proteins were tested using an enzyme-linked immunosorbent assay. With this assay we assessed the binding of TS in solution to proteins adsorbed to polystyrene microtiter plates. Among collagens, platelet TS bound to type V but not to types I, III, or IV. This selective interaction was confirmed in experiments using proteins linked to cyanogen bromide-activated Sepharose. TS released from platelets in response to thrombin activation, as well as that secreted by endothelial cells in culture, bound to type V but not to type I collagen-Sepharose. No binding was observed to denatured type V collagen-Sepharose. The binding region for type V collagen was located in a chymotrypsin-produced fragment of TS with chains of Mr = 70,000, after reduction. Interactions of TS with a number of other proteins, including fibronectin, fibrinogen, and laminin, could be demonstrated using the enzyme-linked immunosorbent assay technique but the interpretation of these findings is difficult since comparable binding to protein-Sepharose was not always observed. Our findings suggest that both the extravascular distribution and function of TS in vivo may involve an interaction with type V collagen.

Thrombospondin: synthesis and secretion by cells in culture.

Thrombospondin, a high molecular weight glycoprotein secreted by platelets in response to activation by thrombin, has been identified by immunofluorescence in bovine aortic endothelial cells, human foreskin fibroblasts, and human aortic smooth muscle cells. Immunofluorescence patterns were found to be similar using antisera raised to thrombospondins purified either from bovine aortic endothelial cells or from human platelets. Radioimmune precipitation of pulse-labeled cellular proteins confirmed the presence of thrombospondin in positively stained cells. A sensitive quantitative enzyme-linked immunosorbent assay (ELISA) was developed and used to determine that the accumulation of secreted thrombospondin was similar for endothelial cells and fibroblasts but was higher for smooth muscle cells. The presence of thrombospondin in a variety of cells suggests that its function may not be limited to an involvement in platelet interactions.

Purification and characterization of smooth muscle cell caveolae.

Plasmalemmal caveolae are a membrane specialization that mediates transcytosis across endothelial cells and the uptake of small molecules and ions by both epithelial and connective tissue cells. Recent findings suggest that caveolae may, in addition, be involved in signal transduction. To better understand the molecular composition of this membrane specialization, we have developed a biochemical method for purifying caveolae from chicken smooth muscle cells. Biochemical and morphological markers indicate that we can obtain approximately 1.5 mg of protein in the caveolae fraction from approximately 100 g of chicken gizzard. Gel electrophoresis shows that there are more than 30 proteins enriched in caveolae relative to the plasma membrane. Among these proteins are: caveolin, a structural molecule of the caveolae coat; multiple, glycosylphosphatidylinositol-anchored membrane proteins; both G alpha and G beta subunits of heterotrimeric GTP-binding protein; and the Ras-related GTP-binding protein, Rap1A/B. The method we have developed will facilitate future studies on the structure and function of caveolae.

Molecular cloning of complementary DNA for the alpha subunit of the G protein that stimulates adenylate cyclase.

A complementary DNA clone encoding the alpha subunit of the adenylate cyclase stimulatory G protein (Gs) was isolated and identified. A bovine brain complementary DNA library was screened with an oligonucleotide probe derived from amino acid sequence common to known G proteins. The only clone that was obtained with this probe has a complementary DNA insert of approximately 1670 base pairs. An antibody to a peptide synthesized according to deduced amino acid sequence reacts specifically with the alpha subunit of Gs. In addition, RNA that hybridizes with probes made from the clone is detected in wild-type S49 cells; however, cyc- S49 cells, which are deficient in Gs alpha activity, are devoid of this messenger RNA.

Influence of gamma subunit prenylation on association of guanine nucleotide-binding regulatory proteins with membranes.

Two approaches were taken to address the possible role of gamma-subunit prenylation in dictating the cellular distribution of guanine nucleotide-binding regulatory proteins. Prenylation of gamma subunits was prevented by site-directed mutagenesis or by inhibiting the synthesis of mevalonate, the precursor of cellular isoprenoids. When beta or gamma subunits were transiently expressed in COS-M6 simian kidney cells (COS) cells, the proteins were found in the membrane fraction by immunoblotting. Immunofluorescence experiments indicated that the proteins were distributed to intracellular structures in addition to plasma membranes. Replacement of Cys68 of gamma with Ser prevented prenylation of the mutant protein and association of the protein with the membrane fraction of COS cells. Immunoblotting results demonstrated that some of the beta subunits were found in the cytoplasm when coexpressed with the nonprenylated mutant gamma subunit. When Neuro 2A cells were treated with compactin to inhibit protein prenylation, a fraction of endogenous beta and gamma was distributed in the cytoplasm. It is concluded that prenylation facilitates association of gamma subunits with membranes, that the cellular location of gamma influences the distribution of beta, and that prenylation is not an absolute requirement for interaction of beta and gamma.

Organization of G proteins and adenylyl cyclase at the plasma membrane.

There is mounting evidence for the organization and compartmentation of signaling molecules at the plasma membrane. We find that hormone-sensitive adenylyl cyclase activity is enriched in a subset of regulatory G protein-containing fractions of the plasma membrane. These subfractions resemble, in low buoyant density, structures of the plasma membrane termed caveolae. Immunofluorescence experiments revealed a punctate pattern of G protein alpha and beta subunits, consistent with concentration of these proteins at distinct sites on the plasma membrane. Partial coincidence of localization of G protein alpha subunits with caveolin (a marker for caveolae) was observed by double immunofluorescence. Results of immunogold electron microscopy suggest that some G protein is associated with invaginated caveolae, but most of the protein resides in irregular structures of the plasma membrane that could not be identified morphologically. Because regulated adenylyl cyclase activity is present in low-density subfractions of plasma membrane from a cell type (S49 lymphoma) that does not express caveolin, this protein is not required for organization of the adenylyl cyclase system. The data suggest that hormone-sensitive adenylyl cyclase systems are localized in a specialized subdomain of the plasma membrane that may optimize the efficiency and fidelity of signal transduction.

Light microscopic immunolocation of thrombospondin in human tissues.

Affinity-purified antisera against thrombospondin were used to locate the presence of this glycoprotein in frozen sections of several human tissues by immunofluorescence techniques. Immunostaining was observed in the peritubular connective tissue and in basement membrane regions beneath glandular epithelium in skin and lung. Intense immunostaining was observed at the dermal-epidermal junction in skin and in small blood vessels throughout this tissue. Skeletal muscle exhibited positive staining with anti-thrombospondin antisera within interstitial areas. Immunostaining was confined to the luminal portions of large blood vessels such as aorta. In large blood vessels that contained lesions of atherosclerosis, immunostaining was observed throughout the lesion area and was especially prominent surrounding some of the lesion cells. These results indicate that thrombospondin is located within the matrix of a variety of human tissues and supports the suggestion that this glycoprotein is an endogenous component of some extracellular matrices.

G proteins in Aplysia: biochemical characterization and regional and subcellular distribution.

We studied G proteins and regulation of adenylate cyclase in nervous tissue and muscle of Aplysia using bacterial toxin-catalyzed ADP-ribosylation. We identified Gs alpha, a Mr 45,000 cholera toxin substrate, Go alpha, a Mr 40,000 pertussis toxin substrate, and G beta (Mr 37,000) by Western blot analysis with antisera specific for bovine brain G protein subunits. Partial proteolysis suggests that the neuronal pertussis toxin substrates are heterogeneous. The concentration of these substrates in membranes from Aplysia ganglia is similar to that of rat, squid and Helix; in Aplysia nervous tissue, G protein subunits are most enriched in synaptosomes and neuropil. The stimulation of adenylate cyclase by serotonin (5-HT), low concentrations of GTP-gamma-S, and cholera toxin, and the inhibition by high concentrations of GTP-gamma-S that is blocked by pertussis toxin indicate that both a Gs and a Gi protein regulate the Aplysia enzyme. These results support the idea that G proteins in Aplysia are important in regulating synaptic function.

Location and partial characterization of the heparin-binding fragment of platelet thrombospondin.

Purified platelet thrombospondin (TS) was subjected to proteolysis with a number of proteases including factors IXa, Xa, thrombin, elastase, trypsin, and chymotrypsin. All enzymes yielded fragments of TS which bound to heparin-Sepharose. Only chymotrypsin cleavage produced a single species of heparin-binding fragment, as analyzed by SDS-PAGE. This fragment had a chain molecular weight of 28,000, and contained no interchain disulfide bonds. Amino acid sequence analysis of the heparin-binding fragment and of TS revealed a single sequence, indicating that the fragment constitutes the amino-terminal domain of TS and that the three chains in TS are identical in this region.

Receptor regulation of G-protein palmitoylation.

Many alpha subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) are palmitoylated. Exposure of cells to the beta-adrenergic agonist isoproterenol increased incorporation of [3H]palmitate specifically into alpha s, the alpha subunit that mediates stimulation of adenylyl cyclase. Pulse-chase experiments suggested that isoproterenol increased turnover of alpha s-bound palmitate. Mutagenesis of Cys-3 in alpha s or alpha o (a homologous alpha subunit) prevented palmitoylation of these proteins. Differing results were obtained when mutations of Cys-3 in alpha s or alpha o were expressed in cells and assayed for their distribution between soluble and membrane fractions. Some alpha subunits, including alpha o, are myristoylated at the amino-terminal glycine residue. Mutation of this glycine prevented both myristoylation and palmitoylation of alpha o, indicating that myristoylation precedes palmitoylation of dually acylated alpha subunits. The amino-terminal sequences and fatty acylation properties of dually acylated alpha subunits are strikingly similar to those of some members of the Src family of protein-tyrosine kinases. The amino-terminal sequence Met-Gly-Cys-Xaa-Xaa-Ser/Cys shared by these proteins may represent a motif for cotranslational and posttranslational processing that includes myristoylation of the glycine residue and reversible palmitoylation of the cysteine residue.

Purification and characterization of the 22,000-dalton GTP-binding protein substrate for ADP-ribosylation by botulinum toxin, G22K.

GTP-binding proteins were purified from human neutrophils, including a 40,000-Da pertussis toxin substrate (Gn) and 22,000-, 24,000-, and 26,000-Da proteins, termed G22K, G24K, and G26K, respectively. The latter proteins were shown to be immunologically unrelated to Gn. G22K cross-reacted with anti-ras monoclonal antibody 142-24EO5, but not with monoclonal antibody Y13-259. A single 22,000-Da substrate for botulinum toxin-catalyzed ADP-ribosylation present in neutrophil membranes co-migrated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis with G22K. In the presence of a cytosolic factor, G22K could serve as a specific botulinum toxin substrate. The 22,000-Da botulinum toxin substrate in neutrophil membranes could be immunoprecipitated by antibody 142-24EO5, but not by antibody Y13-259. G22K appears to be a unique GTP-binding protein which serves as a substrate for ADP-ribosylation by a component of botulinum toxin and which may be involved in exocytotic secretion or cellular differentiation.

Characterization of Gs alpha mRNA transcripts in primary cultures of rat brain astrocytes.

A cDNA clone encoding a stimulatory G-protein alpha subunit (Gs alpha) was isolated from a cDNA library derived from cultured rat astrocytes. The nucleotide sequence of the cDNA indicated that it corresponds to the Gs alpha-2 form of Gs alpha mRNA, one of four Gs alpha mRNAs known to be derived by alternative splicing from the human Gs alpha gene. A ribonuclease protection assay using cRNA from this clone allowed distinction between the Gs alpha-1 and Gs alpha-2 mRNAs, which encode the 52-kDa (Gs-L) forms of Gs alpha. Astrocytes express relatively high amounts of Gs alpha-1 mRNA, much lower amounts of the Gs alpha-2 mRNA, and no detectable amounts of the mRNAs (Gs alpha-3 and Gs alpha-4) encoding the two 45-kDa forms of Gs alpha (Gs alpha-S). Similar results were obtained with RNA samples isolated from whole brain. The 45-kDa form of Gs alpha protein was not detectable by immunoblot analysis of a membrane preparation from rat cerebral cortex (the source of the astrocyte cultures). These results indicate that the expression of Gs alpha forms in astrocytes is similar to that found in whole brain.

Involvement of cyclic GMP in the release of stereotyped behaviour patterns in moths by a peptide hormone.

A peptide hormone, the eclosion hormone, triggers two behavioural patterns--the pre-eclosion and eclosion patterns--when injected into pharate silkmoths. Injection of cyclic nucleotides caused the same behavioural responses with cGMP being 10 to 100 times more potent than cAMP. Exogenous cGMP also acted directly on the isolated nervous system to evoke the characteristic motor programmes. Protection of endogenous cyclic nucleotides by pretreatment of moths with a phosphodiesterase inhibitor, theophylline, markedly enhanced the sensitivity of the moths to the hormone. Injection of partially purified hormone preparations was followed by an increase in nervous system cGMP but not cAMP. The increase preceded the behavioural effectiveness of each dose was correlated with its ability to cause a cGMP increase. It was concluded that the behavioural effects of the eclosion hormone are mediated through an increase in cGMP in the nervous system.

Antisera of designed specificity for subunits of guanine nucleotide-binding regulatory proteins.

Antisera were raised against purified subunits of regulatory GTP-binding proteins (G proteins) and against synthetic peptides that correspond to defined regions of G proteins. Peptide antisera were generated that recognized all alpha or all beta subunits from Gs, Gi, Go, and transducin; others recognized only Gs alpha or Go alpha. Such cross-reaction or complete specificity for a given alpha subunit was not obtained when purified subunits were injected. Peptide antisera were used to identify G protein subunits in selected tissue membrane preparations by immunoblots.

G protein gamma subunits contain a 20-carbon isoprenoid.

A small subset of cellular proteins are covalently modified by the addition of isoprenoid groups. These include p21ras, fungal mating factors, and nuclear lamins, which are isoprenylated at carboxyl-terminal cysteine residues with a 15-carbon farnesyl group. The similarity of the carboxyl-terminal sequences of these proteins with the alpha and gamma subunits of signal-transducing guanine nucleotide-binding regulatory proteins (G proteins) prompted examination of isoprenylation of G protein subunits. PC-12 cells were incubated with the isoprenoid precursor [3H]mevalonolactone. The beta and gamma subunits were isolated by specific association with an affinity column of immobilized alpha subunits. The gamma subunits were radiolabeled, and the tritiated lipid released from them by treatment with methyl iodide comigrated chromatographically with the 20-carbon isoprenoid geranylgeraniol. Label was not detected in G protein alpha or beta subunits. Isoprenylation of gamma subunits by the geranylgeranyl group is presumed to contribute to the association of G proteins with membranes.

G-protein alpha-subunit expression, myristoylation, and membrane association in COS cells.

Myristoylation of seven different alpha subunits of guanine nucleotide-binding regulatory proteins (G proteins) was examined by expressing these proteins in monkey kidney COS cells. Metabolic labeling studies of cells transfected with cytomegalovirus-based expression vectors indicated that [3H]myristate was incorporated into alpha i1, alpha i2, alpha i3, alpha 0, alpha t, and alpha z but not alpha s subunits. The role of myristoylation in the association of alpha subunits with membranes was analyzed by site-directed mutagenesis and by substitution of myristate with a less hydrophobic analog, 10-(propoxy)decanoate (11-oxamyristate). Myristoylation of alpha 0 was blocked when an alanine residue was substituted for its amino-terminal glycine, as was association of the protein with membranes. Substitution of the myristoyl group with 11-oxamyristate affected the cellular distribution of a subset of acylated alpha subunits. The results are consistent with a model wherein the hydrophobic interaction of myristate with the bilayer permits continued association of the protein with the plasma membrane when G-protein alpha subunits dissociate from beta gamma.