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Interaction of two structurally-distinct sequence types with the clathrin terminal domain beta-propellerDrake MT, Traub LM. J Biol Chem 2001 May 29; [epub ahead of print] The amino-terminal domain of the clathrin heavy chain, which folds into a seven-bladed beta-propeller, binds directly to several endocytic proteins via short sequences based on the consensus residues LLDLD. In addition to a single LLDLD-based, type-I clathrin-binding sequence, both amphiphysin and epsin each have a second, distinct sequence that is also capable of binding to clathrin directly. Here, we have analyzed these sequences, which we term type-II sequences, and show that the (257)LMDLA sequence in rat epsin 1 appears to be a weak clathrin-binding variant of the sequence (417)PWDLW originally found in human amphiphysin II. The structural features of the type-II sequence required for association with clathrin are distinct from the LLDLD-based sequence. In the central segment of amphiphysin, thetype-I and type-II sequences cooperate to effect optimal clathrin binding and formation of sedimentable assemblies. Together, the data provide evidence for two interaction surfaces upon certain endocytic accessory proteins that couldcooperate with other coat components to enhance clathrin-bud formation at the cell surface. -----------------------Millard EE, Srivastava K, Traub LM, Schaffer JE, Ory DS. The Niemann-Pick type C1 (NPC1) protein is a key participant in intracellular trafficking of low density lipoproteincholesterol, but its role in regulation of sterol homeostasis is not well understood. To characterize further the function ofNPC1, we generated stable Chinese hamster ovary (CHO) cell lines overexpressing the human NPC1 protein(CHO/NPC1). NPC1 overexpression increases the rate of trafficking of low density lipoprotein cholesterol to theendoplasmic reticulum and the rate of delivery of endosomal cholesterol to the plasma membrane (PM). CHO/NPC1cells exhibit a 1.5-fold increase in total cellular cholesterol and up to a 2.9-fold increase in PM cholesterol. This increasein PM cholesterol is closely paralleled by a 3-fold increase in de novo cholesterol synthesis. Inhibition of cholesterolsynthesis results in marked redistribution of PM cholesterol to intracellular sites, suggesting an unsuspected role forNPC1 in internalization of PM cholesterol. Despite elevated total cellular cholesterol, CHO/NPC1 cells exhibit increased cholesterol synthesis, which may be attributable to both resistance to oxysterol suppression of sterol-regulatedgene expression and to reduced endoplasmic reticulum cholesterol levels under basal conditions. Taken together, thesestudies provide important new insights into the role of NPC1 in the determination of the levels and distribution of cellular cholesterol. -----------------------Sorting in the endosomal system in yeast and animal cells.Lemmon SK, Traub LM. Curr Opin Cell Biol 2000 Aug;12(4):457-66 The endosomal system is a major membrane-sorting apparatus. New evidence reveals that novel coat proteins assistspecific sorting steps and docking factors ensure the vectorial nature of trafficking in the endosomal compartment.There is also good evidence for ubiquitin regulating passage of certain proteins into multivesicular late endosomes,which mature by accumulating invaginated membrane. Lipids play a central role in this involution process, as do the class E vacuolar protein-sorting proteins. -----------------------Epsin binds to clathrin by associating directly with the clathrin-terminal domain. Evidence for cooperative binding through two discrete sitesDrake MT, Downs MA, Traub LM. J Biol Chem 2000 Mar 3;275(9):6479-89 Epsin is a recently identified protein that appears to play an important role in clathrin-mediated endocytosis. The central region of epsin 1, the so-called DPW domain, binds to the heterotetrameric AP-2 adaptor complex by associating directly with the globular appendage of the alpha subunit. We have found that this central portion of epsin 1 also associates with clathrin. The interaction with clathrin is direct and not mediated by epsin-bound AP-2. Alanine scanning mutagenesis shows that clathrin binding depends on the sequence (257)LMDLADV located within the epsin 1 DPW domain. This sequence, related to the known clathrin-binding sequences in the adaptor beta subunits, amphiphysin, and beta-arrestin, facilitates the association of epsin 1 with the terminal domain of the clathrin heavy chain. Unexpectedly, inhibiting the binding of AP-2 to the GST-epsin DPW fusion protein by progressively deleting DPW triplets but leaving the LMDLADV sequence intact, diminishes the association of clathrin in parallel with AP-2. Because the beta subunit of the AP-2 complex also contains a clathrin-binding site, optimal association with soluble clathrin appears to depend on the presence of at least two distinct clathrin-binding sites, and we show that a second clathrin-binding sequence (480)LVDLD, located within the carboxyl-terminal segment of epsin 1, also interacts with clathrin directly. The LMDLADV and LVDLD sequences act cooperatively in clathrin recruitment assays, suggesting that they bind to different sites on the clathrin-terminal domain. The evolutionary conservation of similar clathrin-binding sequences in several metazoan epsin-like molecules suggests that the ability to establish multiple protein-protein contacts within a developing clathrin-coated bud is an important aspect of epsin function. -----------------------Crystal structure of the alpha appendage of AP-2 reveals a recruitment platform for clathrin-coat assemblyTraub LM, Downs MA, Westrich JL, Fremont DH. AP-2 adaptors regulate clathrin-bud formation at the cell surface by recruiting clathrin trimers to the plasma membrane and by selecting certain membrane proteins for inclusion within the developing clathrin-coat structure. These functions are performed by discrete subunits of the adaptor heterotetramer. The carboxyl-terminal appendage of the AP-2 alpha subunit appears to regulate the translocation of several endocytic accessory proteins to the bud site. We have determined the crystal structure of the alpha appendage at 1.4-A resolution by multiwavelength anomalous diffraction phasing. It is composed of two distinct structural modules, a beta-sandwich domain and a mixed alpha-beta platform domain. Structure-based mutagenesis shows that alterations to the molecular surface of a highly conserved region on the platform domain differentially affect associations of the appendage with amphiphysin, eps15, epsin, and AP180, revealing a common protein-binding interface. -----------------------Coupled inositide phosphorylation and phospholipase D activation initiates clathrin-coat assembly on lysosomesArneson LS, Kunz J, Anderson RA, Traub LM. J Biol Chem 1999 Jun 18;274(25):17794-805 Adaptors appear to control clathrin-coat assembly by determining the site of lattice polymerization but the nucleating events that target soluble adaptors to an appropriate membrane are poorly understood. Using an in vitro model system that allows AP-2-containing clathrin coats to assemble on lysosomes, we show that adaptor recruitment and coat initiation requires phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) synthesis. PtdIns(4,5)P2 is generated on lysosomes by the sequential action of a lysosome-associated type II phosphatidylinositol 4-kinase and a soluble type I phosphatidylinositol 4-phosphate 5-kinase. Phosphatidic acid, which potently stimulates type I phosphatidylinositol 4-phosphate 5-kinase activity, is generated on the bilayer by a phospholipase D1-like enzyme located on the lysosomal surface. Quenching phosphatidic acid function with primary alcohols prevents the synthesis of PtdIns(4, 5)P2 and blocks coat assembly. Generating phosphatidic acid directly on lysosomes with exogenous bacterial phospholipase D in the absence of ATP still drives adaptor recruitment and limited coat assembly, indicating that PtdIns(4,5)P2 functions, at least in part, to activate the PtdIns(4,5)P2-dependent phospholipase D1. These results provide the first direct evidence for the involvement of anionic phospholipids in clathrin-coat assembly on membranes and define the enzymes responsible for the production of these important lipid mediators. -----------------------High-affinity binding of the AP-1 adaptor complex to trans-golgi network membranes devoid of mannose 6-phosphate receptorsZhu Y, Traub LM, Kornfeld S. Mol Biol Cell 1999 Mar;10(3):537-49 The GTP-binding protein ADP-ribosylation factor (ARF) initiates clathrin-coat assembly at the trans-Goli network (TGN) by generating high-affinity membrane-binding sites for the AP-1 adaptor complex. Both transmembrane proteins, which are sorted into the assembling coated bud, and novel docking proteins have been suggested to be partners with GTP-bound ARF in generating the AP-1-docking sites. The best characterized, and probably the major transmembrane molecules sorted into the clathrin-coated vesicles that form on the TGN, are the mannose 6-phosphate receptors (MPRs). Here, we have examined the role of the MPRs in the AP-1 recruitment process by comparing fibroblasts derived from embryos of either normal or MPR-negative animals. Despite major alterations to the lysosome compartment in the MPR-deficient cells, the steady-state distribution of AP-1 at the TGN is comparable to that of normal cells. Golgi-enriched membranes prepared from the receptor-negative cells also display an apparently normal capacity to recruit AP-1 in vitro in the presence of ARF and either GTP or GTPgammaS. The AP-1 adaptor is recruited specifically onto the TGN and not onto the numerous abnormal membrane elements that accumulate within the MPR-negative fibroblasts. AP-1 bound to TGN membranes from either normal or MPR-negative fibroblasts is fully resistant to chemical extraction with 1 M Tris-HCl, pH 7, indicating that the adaptor binds to both membrane types with high affinity. The only difference we do note between the Golgi prepared from the MPR-deficient cells and the normal cells is that AP-1 recruited onto the receptor-lacking membranes in the presence of ARF1.GTP is consistently more resistant to extraction with Tris. Because sensitivity to Tris extraction correlates well with nucleotide hydrolysis, this finding might suggest a possible link between MPR sorting and ARF GAP regulation. We conclude that the MPRs are not essential determinants in the initial steps of AP-1 binding to the TGN but, instead, they may play a regulatory role in clathrin-coated vesicle formation by affecting ARF.GTP hydrolysis. -----------------------ADP-ribosylation factor 1 transiently activates high-affinity adaptor protein complex AP-1 binding sites on Golgi membranesZhu Y, Traub LM, Kornfeld S. Mol Biol Cell 1998 Jun;9(6):1323-37 Association of the Golgi-specific adaptor protein complex 1 (AP-1) with the membrane is a prerequisite for clathrin coat assembly on the trans-Golgi network (TGN). The AP-1 adaptor is efficiently recruited from cytosol onto the TGN by myristoylated ADP-ribosylation factor 1 (ARF1) in the presence of the poorly hydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPS). Substituting GTP for GTPS, however, results in only poor AP-1 binding. Here we show that both AP-1 and clathrin can be recruited efficiently onto the TGN in the presence of GTP when cytosol is supplemented with ARF1. Optimal recruitment occurs at 4 µM ARF1 and with 1 mM GTP. The AP-1 recruited by ARF1·GTP is released from the Golgi membrane by treatment with 1 M Tris-HCl (pH 7) or upon reincubation at 37°C, whereas AP-1 recruited with GTPS or by a constitutively active point mutant, ARF1(Q71L), remains membrane bound after either treatment. An incubation performed with added ARF1, GTP, and AlFn, used to block ARF GTPase-activating protein activity, results in membrane-associated AP-1, which is largely insensitive to Tris extraction. Thus, ARF1·GTP hydrolysis results in lower-affinity binding of AP-1 to the TGN. Using two-stage assays in which ARF1·GTP first primes the Golgi membrane at 37°C, followed by AP-1 binding on ice, we find that the high-affinity nucleating sites generated in the priming stage are rapidly lost. In addition, the AP-1 bound to primed Golgi membranes during a second-stage incubation on ice is fully sensitive to Tris extraction, indicating that the priming stage has passed the ARF1·GTP hydrolysis point. Thus, hydrolysis of ARF1·GTP at the priming sites can occur even before AP-1 binding. Our finding that purified clathrin-coated vesicles contain little ARF1 supports the concept that ARF1 functions in the coat assembly process rather than during the vesicle-uncoating step. We conclude that ARF1 is a limiting factor in the GTP-stimulated recruitment of AP-1 in vitro and that it appears to function in a stoichiometric manner to generate high-affinity AP-1 binding sites that have a relatively short half-life. -----------------------The trans-Golgi network: a late secretory sorting stationTraub LM, Kornfeld S. Curr Opin Cell Biol 1997 Aug;9(4):527-33 Proteins synthesized on membrane-bound ribosomes are transported through the Golgi apparatus and, on reaching the trans-Golgi network, are sorted for delivery to various cellular destinations. Sorting involves the assembly of cytosol-oriented coat structures which preferentially package cargo into vesicular transport intermediates. Recent studies have shed new light on both the molecular machinery involved and the complexity of the sorting processes. -----------------------AP-2-containing clathrin coats assemble on mature lysosomes.Traub LM, Bannykh SI, Rodel JE, Aridor M, Balch WE, Kornfeld S. Coat proteins appear to play a general role in intracellular protein trafficking by coordinating a membrane budding event with cargo selection. Here we show that the AP-2 adaptor, a clathrin-associated coat-protein complex that nucleates clathrin-coated vesicle formation at the cell surface, can also initiate the assembly of normal polyhedral clathrin coats on dense lysosomes under physiological conditions in vitro. Clathrin coat formation on lysosomes is temperature dependent, displays an absolute requirement for ATP, and occurs in both semi-intact cells and onpurified lysosomes, suggesting that clathrin-coated vesicles might regulate retrograde membrane traffic out of the lysosomal compartment. -----------------------Different domains of the AP-1 adaptor complex are required for Golgi membrane binding and clathrin recruitmentTraub LM, Kornfeld S, Ungewickell E. J Biol Chem 1995 Mar 3;270(9):4933-42 The assembly of clathrin-coated buds on the Golgi requires the recruitment of the heterotetrameric AP-1 adaptor complex, which is dependent on both guanine nucleotides and the small GTP-binding protein ADP-ribosylation factor (ARF). Here, we have investigated the structural domains of the AP-1 complex necessary for ARF-mediated translocation of the adaptor complex onto Golgi membranes and the subsequent recruitment of clathrin onto the membrane. Controlled proteolysis of purified AP-1, derived from bovine adrenal coated vesicles, was used to generate AP-1 core fragments composed of the amino-terminal trunk regions of the beta 1 and gamma subunits and associated mu 1 and sigma 1 subunits, and lacking either the beta 1 subunit carboxyl-terminal appendage or both beta 1 and gamma subunit appendages. On addition of these truncated fragments to AP-1-depleted adrenal cytosol, both types of core fragments were efficiently recruited onto Golgi membranes in the presence of GTP gamma S. Recruitment of both core fragments was inhibited by the fungal metabolite brefeldin A, indicative of an ARF-dependent process. Limited tryptic digestion of recruited, intact cytosolic AP-1 resulted in the quantitative release of the globular carboxyl-terminal appendage domains of the beta 1 and gamma subunits. The adaptor core complex remained associated with the Golgi membranes. Recruitment of cytosolic clathrin onto the Golgi membranes was strictly dependent on the presence of intact AP-1. Tryptic removal of the beta 1 subunit appendage prevented subsequent clathrin recruitment. We conclude that the structural determinants required for the ARF-mediated binding of cytosolic AP-1 onto Golgi membranes are contained within the adaptor core, and that the carboxyl-terminal appendage domains of the beta 1 and gamma subunits do not play any role in this process. Subsequent recruitment of cytosolic clathrin, however, requires an intact beta 1 subunit. -----------------------Biochemical dissection of AP-1 recruitment onto Golgi membranesTraub LM, Ostrom JA, Kornfeld S. J Cell Biol 1993 Nov;123(3):561-73 Recruitment of the Golgi-specific AP-1 adaptor complex onto Golgi membranes is thought to be a prerequisite for clathrin coat assembly on the TGN. We have used an in vitro assay to examine the translocation of cytosolic AP-1 onto purified Golgi membranes. Association of AP-1 with the membranes required GTP or GTP analogues and was inhibited by the fungal metabolite, brefeldin A. In the presence of GTP gamma S, binding of AP-1 to Golgi membranes was strictly dependent on the concentration of cytosol added to the assay. AP-1 recruitment was also found to be temperature dependent, and relatively rapid at 37 degrees C, following a lag period of 3 to 4 min. Using only an adaptor-enriched fraction from cytosol, purified myristoylated ARF1, and Golgi membranes, the GTP gamma S-dependent recruitment of AP-1 could be reconstituted. Our results show that the association of the AP-1 complex with Golgi membranes, like the coatomer complex, requires ARF, which accounts for the sensitivity of both to brefeldin A. In addition, they provide the basis for a model for the early biochemical events that lead to clathrin-coated vesicle formation on the TGN. -----------------------Hulkower KI, Sagi-Eisenberg R, Traub LM, Georgescu HI, Evans CH. Eur J Biochem Lapine synovial fibroblasts produce prostaglandin E2 (PGE2) and neutral metalloproteinases in response to phorbol 12-myristate 13-acetate (PMA), human recombinant interleukin-1 (hrIL-1) and, in an autocrine fashion, in response to partially purified preparations of their own cytokines known as cell-activating factors (CAF). Here we have examined the possible role of protein kinase C (PKC) in these responses. Whereas the 80-kDa substrate for PKC could not be detected in synovial fibroblasts, these cells contained a 35-kDa protein which fulfilled the criteria for qualifying as a specific substrate of PKC. Translocation assays based upon phosphorylation of the 35-kDa protein and Western blotting techniques allowed the movement of PKC from the cytosolic to the particulate fraction in response to PMA and CAF to be detected but not in response to 4 alpha-PMA or hrIL-1. Inhibitors of PKC suppressed synovial activation by PMA, partially blocked activation by CAF but had no effect on activation by hrIL-1. There thus appear to be PKC-dependent and PKC-independent routes to synovial cell activation. Our data suggest that IL-1 uses the latter, while CAF contains cytokines which utilize both routes. -----------------------Purification of p100, a protein antigenically related to the signal transducing G proteins Gt and Gi. Evidence for an adaptin-like proteinTraub LM, Sagi-Eisenberg R. J Biol Chem 1991 Dec 25;266(36):24642-9 A 100-kDa protein, termed p100, cross-reacts with antisera raised against a synthetic peptide corresponding to the carboxyl-terminal decapeptide of the alpha-subunit of the retinal G protein Gt. p100 is abundantly expressed in liver and, on subcellular fractionation of rat liver homogenates, is distributed between the cytosolic and microsome fractions (Traub, L. M., Evans, W. H., and Sagi-Eisenberg, R. (1990) Biochem. J. 272, 453-458; Udrisar, D., and Rodbell, M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6321-6325). We have now purified p100 to near-homogeneity from rat liver microsomes. The protein was purified approximately 500-fold by ATP extraction followed by a series of four chromatographic steps. Similar to partially purified p100, on two-dimensional electrophoresis, the final preparation contained a major series of five immunoreactive 100-kDa charge isoforms. Partial amino terminus amino acid sequencing of the purified protein revealed that p100 is a previously unidentified protein. Further analysis of the soluble form of p100 showed the protein migrated with an apparent molecular weight of approximately 110,000 on gel filtration, indicating that the soluble protein occurs as a monomeric polypeptide. The soluble form of p100 was also partially purified from rat liver cytosol and amino acid sequencing yielded the same amino-terminal sequence as obtained from the microsome-associated form. The amino-terminal sequence of p100 exhibits significant similarity to the deduced amino-terminal amino acid sequences of both alpha- and gamma-adaptins. Using the amino-terminal sequence from p100, we have raised antipeptide polyclonal antisera. The antisera reacted specifically with the purified 100-kDa protein on immunoblots. With the purified protein and specific antisera now available, it will be possible to explore the physiological role of p100. -----------------------Characterization of the interaction between p100, a novel G-protein-related protein, and rat liver endosomes.Traub LM, Shai E, Sagi-Eisenberg R. Biochem J 1991 Nov 15;280 ( Pt 1):171-8 p100 is a recently identified 100 kDa protein which shares a putative receptor-binding sequence with the signal transducing G-proteins Gt and Gi. In liver, p100 immunoreactivity is distributed between the cytosolic and the microsomal fractions [Traub, Evans & Sagi-Eisenberg (1990) Biochem. J. 272, 453-458; Udrisar & Rodbell (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6321-6325]. More specifically, we have localized the membrane-associated form of p100 to an endosomal subfraction of rat liver microsomes. In this study we have investigated the nature of the interaction between p100 and microsomal membranes. p100 was located on the cytoplasmic surface of the microsomal vesicles, and could be released by treatment with 0.5 M-NaCl or 0.5 M-Tris/HCl, pH 7.0. However, p100 was not released by non-ionic detergents, such as Triton X-100. Binding of p100 to the membrane was reversible, as both membrane-released and cytosolic p100 could re-bind stripped (Tris-washed) microsomes. Soluble p100 could not, however, bind to untreated microsomes. Binding to stripped microsomes approached saturation and was inhibited by up to 60% by either heat treatment or mild trypsin treatment of the vesicles. This implies that the interaction between p100 and the microsomal vesicles involves the direct binding of p100 to vesicular proteins. This binding was regulated by both adenine and guanine nucleotides. As p100 contains a region similar to the C-terminal decapeptide of alpha i, (the alpha-subunit of Gi) and has a localization that is restricted to an endosomal subfraction, we propose that cytosolic p100 may bind to cytoplasmically exposed domains of internalized receptors. Thus, like the adaptins, p100 may be involved in the process of sorting and receptor trafficking through the endosomal compartment of the cells. -----------------------Interleukin-1 and synovial protein kinase C: identification of a novel, 35 kDa cytosolic substrate.Hulkower KI, Sagi-Eisenberg R, Traub LM, Georgescu HI, Evans CH. We have been examining the role of protein kinase C (PKC) in synovial cell activation in response to interleukin-1 (IL-1). Attempts to measure PKC in soluble extracts of synovial fibroblasts by standard techniques failed. Western blotting with anti-PKC antibodies detected only a low level of PKC in synovial cells compared to rat basophilic leukemia cells and crude brain extracts. However, synovial PKC could be detected by measuring the Ca(2+)- and phospholipid-dependent phosphorylation of endogenous substrates. In this way, a 35 kDa protein was identified as the major endogenous cytosolic substrate for PKC. Treatment of synoviocytes with phorbol myristate acetate (PMA) strongly induced the synthesis of neutral metalloproteinases (NPs) and prostaglandin E2 (PGE2). Both Western blotting and assays based upon phosphorylation of the 35 kDa protein confirmed translocation of PKC from the cytosol in response to PMA. Although IL-1 induced the NPs and PGE2, it did so without detectable translocation of PKC. There thus appear to be PKC-dependent and PKC-independent routes of synovial cell activation. Our data suggest that IL-1 uses the latter. -----------------------A novel 100 kDa protein, localized to receptor-enriched endosomes, is immunologically related to the signal-transducing guanine-nucleotide-binding proteins Gt and GiTraub LM, Evans WH, Sagi-Eisenberg R. Biochem J 1990 Dec 1;272(2):453-8 Antisera raised against the C-terminus decapeptide of the alpha-subunit of the retinal guanine-nucleotide-binding protein (G-protein) transducing (Gt) cross-reacted with the alpha-subunit of the inhibitory G-protein Gi. The same antisera also reacted with a 100 kDa protein (p100) found in rat liver homogenates. The immunoreactivity of both Gt and p100 was specifically inhibited by the immunizing peptide with similar dose-dependencies [concn. causing 50% inhibition (IC50) = 300 ng/ml]. This similarity in inhibition profiles implies that p100 contains within its structure the C-terminal sequence shared by both alpha t and alpha i. Tissue distribution studies demonstrated that p100 was particularly enriched in the liver and kidney, but was also present in other rat tissues, as well as in a number of cell lines tested. In the liver, p100 was found in both the soluble and membrane fractions. The membrane-associated form of p100 was specifically localized to an endosomal fraction (termed D-R), previously shown to be a ligand-free but receptor-enriched subfraction of liver endosomal vesicles. Two-dimensional gel electrophoresis revealed that both the cytosolic and membrane-bound forms of p100 occurred as a series of 100 kDa polypeptides with considerable charge heterogeneity (pI 6-7). Because the C-terminus domains of both alpha t and alpha i facilitate their association with their respective receptors, this region has been functionally assigned as the receptor binding site. Therefore the presence of an immunologically similar region within p100, together with its localization to the receptor-rich endocytic vesicles, suggests that p100 may be a receptor binding protein involved in receptor trafficking. -----------------------Exocytosis in mast cells by basic secretagogues: evidence for direct activation of GTP-binding proteinsAridor M, Traub LM, Sagi-Eisenberg R. J Cell Biol 1990 Sep;111(3):909-17 Histamine release induced by the introduction of a nonhydrolyzable analogue of GTP, GTP-gamma-S, into ATP-permeabilized mast cells, is associated with phosphoinositide breakdown, as evidenced by the production of phosphatidic acid (PA) in a neomycin-sensitive process. The dependency of both PA formation and histamine secretion on GTP-gamma-S concentrations is bell shaped. Whereas concentrations of up to 0.1 mM GTP-gamma-S stimulate both processes, at higher concentrations the cells' responsiveness is inhibited. At a concentration of 1 mM, GTP-gamma-S self-inhibits both PA formation and histamine secretion. Inhibition of secretion can, however, be overcome by the basic secretagogues compound 48/80 and mastoparan that in suboptimal doses synergize with 1 mM GTP-gamma-S to potentiate secretion. Secretion under these conditions is not accompanied by PA formation and is resistant both to depletion of Ca2+ from internal stores and to pertussis toxin (PtX) treatment. In addition, 48/80, like mastoparan, is capable of directly stimulating the GTPase activity of G-proteins in a cell-free system. Together, our results are consistent with a model in which the continuous activation of a phosphoinositide-hydrolyzing phospholipase C (PLC) by a stimulatory G-protein suffices to trigger histamine secretion. Basic secretagogues of mast cells, such as compound 48/80 and mastoparan, are capable of inducing secretion in a mechanism that bypasses PLC by directly activating a G-protein that is presumably located downstream from PLC (GE). Thereby, these secretagogues induce histamine secretion in a receptor-independent manner. -----------------------Protein kinase C-mediated phosphorylation of retinal rod outer segment membrane proteinsSagi-Eisenberg R, Traub LM, Spiegel AM, Zick Y. Cell Signal 1989;1(5):519-31 We have previously reported that the purified GDP-bound alpha-subunit of the GTP-binding protein transducin (TD), present in outer segments of retinal rod cells (ROS), serves as a high affinity substrate (Km = 1 microM) for protein kinase C (PKC) [Zick et al. (1986) Proc. natn. Acad. Sci., U.S.A. 83, 9294-9297]. In the present study we demonstrate that TD-alpha undergoes phosphorylation by PKC when present in its native form in intact ROS membranes. This phosphorylation is inhibited by GTP-gamma-S which activates TD, suggesting that it is only the inactive conformation of TD-alpha that serves as a substrate for PKC. Indeed, both vanadate and AlF4, that confer an active conformation on TD-alpha-GDP, inhibit PKC-mediated phosphorylation of purified TD-alpha-GDP. We demonstrate that the purified beta subunit of TD also serves as an in vitro substrate for PKC. Moreover, following their phosphorylation, both TD-alpha and beta form high affinity complexes with PKC. This is evident from the findings that PKC coprecipitates with both the alpha and beta subunits of TD when the latter are immunoprecipitated by their respective antibodies. PKC phosphorylates additional ROS proteins of 36, 48 and 92 kDa, tentatively identified as rhodopsin, arrestin and the cGMP-phosphodiesterase. Taken together our results strongly suggest that phosphorylation of TD is of physiological relevance and that through phosphorylation of endogenous ROS proteins, PKC could play a key role in regulating phototransduction.
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