A novel microtubule-associated protein from mammalian nerve shows ATP- sensitive binding to microtubules

We report the isolation of a protein from mammalian nerve which shows ATP-sensitive binding to microtubules and ATPase activity. This protein, which we have designated HMW4, was prepared from bovine spinal nerve roots by microtubule affinity and ATP-induced release, and was further purified by sucrose density gradient centrifugation. It is a high molecular weight protein with a denatured Mr of 315,000, a Stokes radius of 90 A, and a sedimentation value of approximately 19S. It can be resolved electrophoretically from the well-characterized bovine brain microtubule-associated proteins (MAPs) and also appears to be distinct from MAP 1C. HMW4 has a vanadate-sensitive and azide-insensitive ATPase activity which averages 20 nmol Pi/min per mg protein and is different from dynein and myosin ATPases. HMW4 prepared on sucrose gradients exhibits binding to MAP-free microtubules in the absence of ATP which is reduced by ATP addition. Assayed by darkfield microscopy, HMW4 causes bundling of MAP-free microtubules which is reversed by ATP addition.     

Stopped-flow measurement of cytoskeletal contraction: Dictyostelium myosin II is specifically required for contraction of amoeba cytoskeletons

Cytoskeletons provide valuable information on the composition and organization of the cell's contractile machinery, and in many cases these cell models retain the ability to contract. To quantitate contraction rates, we developed a novel stopped-flow assay permitting simultaneous analysis of thousands of Dictyostelium cytoskeletons within milliseconds of mixing with Mg-ATP. Cytoskeletons were placed in one syringe of the stopped flow apparatus and the appropriate buffer was placed in the second syringe. Mixing with Mg-ATP caused an immediate increase in the absorbance at 310 nm. Rapid fixation of the cytoskeletons during the reaction confirmed that this change in absorbance was highly correlated with contraction of the cytoskeletons. This spectroscopic change was used to measure the effects of temperature, pH, ionic strength, and nucleotides on contraction rate. Treatment with high salt and ATP removed most of the myosin, some actin, and small amounts of minor proteins. These extracted cytoskeletons lost the ability to contract, but after the addition of purified Dictyostelium myosin they regained full function. In contrast, rabbit skeletal muscle myosin was unable to restore contractility, even though it bound to the extracted cytoskeletons. Cytoskeletons prepared from a myosin-null mutant did not contract. Upon the addition of purified ameba myosin, however, they became contractile. These results suggest that filamentous Dictyostelium myosin II is essential for contraction, and that the actin cytoskeleton and associated proteins retain their functional organization in the absence of myosin.     

Isolation and characterization of protein kinase C from Y-1 adrenal cell cytoskeleton

The cytoskeletons of Y-1 mouse adrenal tumor cells contain a calcium and phospholipid-dependent protein kinase (protein kinase C) that is bound sufficiently tight to resist extraction by 0.5% Triton but not by 1.0% Triton. The enzyme has been purified to near homogeneity from cytoskeleton and cytosol. It shows features typical of this type of kinase, namely a requirement for Ca2+ and phospholipid, stimulation by tumor promoters but not by nontumor-promoting phorbol esters, and inhibition by trifluoperazine. The enzyme shows specificity for four substrates found in the cytoskeleton, namely 80, 33, 20, and 18 kD. The first three substrates are phosphorylated by the enzyme; the fourth is dephosphorylated and is therefore affected by the kinase indirectly. The 80-kD protein is the kinase enzyme itself which is autophosphorylated in vitro and in the cytoskeleton. The 20-kD protein is myosin light chain. The 33- and 18-kD proteins are unidentified. The same substrates were phosphorylated when Y-1 cells were permeabilized with digitonin and incubated with [gamma-32P]ATP and phorbol-12-myristate-13-acetate. Partly purified protein kinase C changes the extent of phosphorylation of the same substrates when added to cytoskeletons previously extracted to remove endogenous protein kinase C. Addition of Ca2+, phosphatidylserine, and phorbol-12-myristate-13-acetate to cytoskeletons, and addition of these three agents plus protein kinase C to extracted cytoskeletons, causes these structures to undergo a rapid and extensive rounding. A similar change is induced in intact cells by addition of phorbol ester. It is concluded that protein kinase C is capable of changing the shape of adrenal cells by an action that involves autophosphorylation and phosphorylation of myosin light chain. This response may in turn be related to the steroidogenic responses to ACTH and cyclic AMP.     

Changes in the association of actin-binding proteins with the actin cytoskeleton during chemotactic stimulation of Dictyostelium discoideum

Triton-insoluble cytoskeletons were isolated from Dictyostelium discoideum AX3 cells prior to and following stimulation with 2'deoxy cyclic adenosine monophosphate (cAMP). Temporal changes in the content of actin and a 120,000 dalton actin-binding protein (ABP-120) in cytoskeletons following stimulation were monitored. Both actin and ABP-120 were incorporated into the cytoskeleton at 30-40 seconds following stimulation, which is cotemporal with the onset of pseudopod extension during stimulation of amoebae with chemoattractants. Changes in the content of total cytoskeletal protein and cytoskeletal myosin were determined under the same experimental conditions as controls. These proteins exhibited different kinetics from those of cytoskeletal ABP-120 and actin following the addition of 2'deoxy cAMP. The authors concluded that the association of ABP-120 with the cytoskeleton is regulated during cAMP signalling. Furthermore, these results indicate that ABP-120 is involved in cross-linking newly assembled actin filaments into the cytoskeleton during chemoattractant-stimulated pseudopod extension.     

Skelemins: cytoskeletal proteins located at the periphery of M-discs in mammalian striated muscle

The cytoskeletons of mammalian striated and smooth muscles contain a pair of high molecular weight (HMW) polypeptides of 220,000 and 200,000 mol wt, each with isoelectric points of about 5 (Price, M. G., 1984, Am. J. Physiol., 246:H566-572) in a molar ratio of 1:1:20 with desmin. The HMW polypeptides of mammalian muscle have been named "skelemins," because they are in the insoluble cytoskeletons of striated muscle and are at the M-discs. I have used two-dimensional peptide mapping to show that the two skelemin polypeptides are closely related to each another. Polyclonal antibodies directed against skelemins were used to demonstrate that they are immunologically distinct from talin, fodrin, myosin heavy chain, synemin, microtubule-associated proteins, and numerous other proteins of similar molecular weight, and are not oligomers of other muscle proteins. Skelemins appear not to be proteolytic products of larger proteins, as shown by immunoautoradiography on 3% polyacrylamide gels. Skelemins are predominantly cytoskeletal, with little extractable from myofibrils by various salt solutions. Human, bovine, and rat cardiac, skeletal, and smooth muscles, but not chicken muscles, contain proteins cross-reacting with anti-skelemin antibodies. Skelemins are localized by immunofluorescence at the M-lines of cardiac and skeletal muscle, in 0.4-micron-wide smooth striations. Cross sections reveal that skelemins are located at the periphery of the M-discs. Skelemins are seen in threads linking isolated myofibrils at the M-discs. There is sufficient skelemin in striated muscle to wrap around the M-disc about three times, if the skelemin molecules are laid end to end, assuming a length-to-weight ratio similar to M-line protein and other elongated proteins. The results indicate that skelemins form linked rings around the periphery of the myofibrillar M-discs. These cytoskeletal rings may play a role in the maintenance of the structural integrity of striated muscle throughout cycles of contraction and relaxation.     

ATP-induced Secretion in PC12 Cells and Photoaffinity Labeling of Receptors

Abstract— Secretion of catecholamines by rat PC12 cells is strongly stimulated by extracellular ATP via a P₂-type pur-inergic receptor. ATP-induced norepinephrine release was inhibited 80% when extracellular Ca²⁺ was absent. Only four nucleotides, ATP, ATPγS, benzoylbenzoyl ATP (BzATP), and 2-methylthio-ATP, gave substantial stimulation of norepinephrine release from PC12 cells. ATP-induced secretion was inhibited by Mg²⁺, and this inhibition was overcome by the addition of excess ATP suggesting that ATP^4-was the active ligand. ATP-induced secretion of catecholamine release was enhanced by treatment of cells with pertussis toxin or 12- O -tetradecanoylphorbol 13-acetate. The stimulatory effects of 12- O -tetradecanoyl-phorbol 13-acetate and pertussis toxin on norepinephrine release were additive. After brief exposure of intact cells to the photoaffinity analog, [α-³²P]BzATP, two major proteins of 44 and 50 kDa and a minor protein of 97 kDa were labeled. An excess of ATP-γS and BzATP but not GTP blocked labeling of the proteins by [³²P]BzATP. Labeling of the 50-kDa protein was more sensitive to competition by 2-methylthio-ATP than the other labeled proteins, suggesting that the 50-kDa protein represents the P₂ receptor responsible for ATP-stimulated secretion in these cells.     

A protein factor from Bufo marinus erythrocytes cross-bridges microtubules in vitro

A microtubule cross-bridging factor was isolated from erythrocytes of the toad, Bufo marinus. Erythrocytes were lysed and their cytoskeletons disassembled by sonication and high salt extraction. The solubilized proteins were recovered and fractionated using Sephadex G-200 column chromatography. The protein fractions from the column were analysed by SDS-PAGE and pooled into three groups: high molecular weight (HMW) proteins that eluted from the column in the void volume and had a protein composition that included HMW polypeptides; intermediate MW proteins that were shown by SDS-PAGE to contain polypeptides smaller than 120,000 D; and low MW (LMW) proteins that contained polypeptides smaller than 70,000 D. Each group was further fractionated by phosphocellulose (PC) chromatography. The flow-through was recovered, and bound proteins were then eluted by a step gradient of salt (0.2, 0.4, 0.6 and 0.8 M KCl). To assay for microtubule cross-bridging activity, column fractions were incubated with taxol-stabilized microtubules, formed from PC-purified brain tubulin (PC microtubules). Negatively stained samples were examined in the electron microscope for the reconstitution of microtubule bundles with interconnecting cross-bridges. The HMW protein fraction from the G-200 column contained the cross-bridging factor. When these proteins were further fractionated by PC chromatography only the fraction eluted by 0.2 M KCl induced the formation of microtubule bundles with cross-bridges. No other protein fraction isolated by the described method revealed cross-bridges between microtubules in vitro.     

Asymmetric interactions of ATP with the AAA+ ClpX6 unfoldase: allosteric control of a protein machine

ATP hydrolysis by AAA+ ClpX hexamers powers protein unfolding and translocation during ClpXP degradation. Although ClpX is a homohexamer, positive and negative allosteric interactions partition six potential nucleotide binding sites into three classes with asymmetric properties. Some sites release ATP rapidly, others release ATP slowly, and at least two sites remain nucleotide free. Recognition of the degradation tag of protein substrates requires ATP binding to one set of sites and ATP or ADP binding to a second set of sites, suggesting a mechanism that allows repeated unfolding attempts without substrate release over multiple ATPase cycles. Our results rule out concerted hydrolysis models involving ClpX(6)*ATP(6) or ClpX(6)*ADP(6) and highlight structures of hexameric AAA+ machines with three or four nucleotides as likely functional states. These studies further emphasize commonalities between distant AAA+ family members, including protein and DNA translocases, helicases, motor proteins, clamp loaders, and other ATP-dependent enzymes.     

Sindbis病毒的蛋白质合成与细胞骨架的关系

In our experiments, protein synthesis of host cells were inhibited quickly at the early stage of infection by Sindbis virus. Polysome and mRNA of host cell fell off from cytoskeletons, whereas virus RNA bound up. We also found it was via 3'-terminal that virus RNA bound with cytoskeleton. After studying on the virus nonstructural proteins, we found the synthesis and processing of virus protein in vitro were far slowly than in vivo, and most of proteins were premature. So, the cytoskeletons may play an important role there. After treated with colchicine and cytochalasin B, the microtubule and microfilament were destroyed. However, the synthesis and processing of nonstructural proteins of Sindbis virus didn't change much, while the structural proteins were inhibited largely. These results showed the differences of dependence of the synthesis of the two kinds of proteins on cytoskeletons. Microtubule and microfilament may be more important to the synthesis of structural proteins than to that of the nonstructural proteins.     

Unfolded proteins stimulate molecular chaperone Hsc70 ATPase by accelerating ADP/ATP exchange.

The mammalian 70-kilodalton heat shock cognate protein (Hsc70) is an abundant, cytosolic molecular chaperone whose interactions with protein substrates are regulated by ATP hydrolysis. In vitro, purified Hsc70 was found to have a slow, intrinsic ATPase activity in the absence of protein substrates. The addition of an unfolded protein such as apocytochrome c stimulated ATP hydrolysis 2-3-fold. In contrast, the native holoprotein, cytochrome c, did not stimulate the ATPase rate, in accord with recent observations that 70-kilodalton heat shock proteins interact selectively with unfolded proteins. Stimulation of ATP hydrolysis by apocytochrome c was due to an increase in the Vmax, with no effect on the Km for ATP. Following hydrolysis of [3H]ATP, a relatively stable [3H]ADP.Hsc70 complex was formed. Release of [3H]ADP from Hsc70 was most efficient in the presence of other nucleotides such as ADP or ATP, suggesting that ADP release occurs as an ADP/ATP exchange reaction. The loss of radiolabeled ADP from Hsc70 in the presence of exogenous nucleotides followed first-order kinetics. In the presence of nucleotides, apocytochrome c induced a 2-fold increase in the rate of ADP release from Hsc70. Moreover, rate constants of the nucleotide exchange reaction measured in the absence and presence of apocytochrome c (0.16 and 0.34 min-1, respectively) closely matched the kcat values derived from ATP hydrolysis measurements (0.15 and 0.38 min-1, respectively). The results suggest that ADP release in a rate-limiting step in the Hsc70 ATPase reaction and that unfolded proteins stimulate ATP hydrolysis by accelerating the rate of ADP/ATP exchange.     

Sindbis病毒的蛋白质合成与细胞骨架的关系

我们以Sindbis病毒感染BHK-21细胞为模式,研究了病毒的感染与细胞骨架的关系。结果显示:在病毒感染早期,细胞的蛋白质合成迅速被抑制,细胞的多聚核糖体(polysome)和mRNA从骨架上脱落,而病毒的RNA结合到骨架上。我们的结果还进一步表明,病毒的RNA是通过其3′-尾端与骨架结合的。另一方面在对Sindbis病毒非结构蛋白在体内与体外合成与加工的比较中,我们发现病毒蛋白在体外翻译加工的速度远低于体内,并且出现很多未成熟蛋白(premature protein),这种区别可能在某种程度上反应细胞骨架在蛋白质合成与加工中的作用。此外,在用秋水仙素和细胞松驰素B破坏微管和微丝后,病毒非结构蛋白的合成与加工没有明显变化,而结构蛋白的合成则受到明显的抑制。这表明病毒的两类蛋白的合成所依赖的细胞骨架成分可能有所不同,在结构蛋白合成过程中,微丝和微管起了重要作用,在非结构蛋白合成过程中,中间丝很可能起了重要作用。     

MIR is a novel ERM-like protein that interacts with myosin regulatory light chain and inhibits neurite outgrowth

The ERM protein family members ezrin, radixin, and moesin are cytoskeletal effector proteins linking actin to membrane-bound proteins at the cell surface. Here we report on the cloning of myosin regulatory light chain interacting protein (MIR), a protein with an ERM-homology domain and a carboxyl-terminal RING finger, that is expressed, among other tissues, in brain. MIR is distributed in cultured COS cells, in a punctuated manner as shown using enhanced green fluorescent protein (EGFP)-tagged MIR and by staining with a specific antibody for MIR. In the yeast two-hybrid system and in transfected COS cells, MIR interacts with myosin regulatory light chain B, which in turn regulates the activity of the actomyosin complex. Overexpression of MIR cDNA in PC12 cells abrogated neurite outgrowth induced by nerve growth factor (NGF) without affecting TrkA signaling. The results show that MIR, a novel ERM-like protein, affects cytoskeleton interactions regulating cell motility, such as neurite outgrowth.     

DNase-I-dependent dissociation of erythrocyte cytoskeletons

The human erythrocyte contains a complex of peripheral membrane proteins which forms an extensive network or cytoskeleton on the cytoplasmic membrane surface. When I treat erythrocyte cytoskeletons with deoxyribonuclease I (DNase I), the cytoskeletons dissociate and erythrocyte actin is solubilized. The dissociation of the cytoskeletons by DNase I parallels the disruption of actin filaments in vitro by DNase I and is blocked by the addition of action to the DNase I. Large protein complexes remain after DNase I disrupts the cytoskeletons, but these complexes are no longer visible in the light microscope nor sedimentable and are selectively depleted with respect to actin. From these studies, I suggest that DNase I binds to and solubilizes actin, which serves as a structural link between protein complexes in the erythrocyte cytoskeleton.     

''Caged cytoskeletons'': a rapid method for the isolation of microtubule-associated proteins from synchronized plant suspension cells

In the cytoskeleton method for isolating microtubule-associated proteins MAP65, DcKRP120-1 and DcKRP120-2, carrot cells are first converted to protoplasts but this method cannot be used to isolate mitotic MAPs as mitotic synchrony is eroded during lengthy cellulase treatment. Anti-microtubule cycle blocks would also be unsuitable. We report here a method for overcoming these problems. Cellulase degradation of tobacco BY-2 cells for only several minutes allows extraction of detergent-soluble proteins, leaving synchronized "caged cytoskeletons" for depolymerization and enabling affinity purification of MAPs on neurotubules. This rapid and simple method should be of general utility: it can be bulked up, avoids anti-microtubule blocks, and is applicable to other cell suspensions. The effectiveness of the caged cytoskeleton method is demonstrated by comparing known MAPs (the 65 kDa structural MAPs and the kinesin-related protein, TKRP125) in synchronized cells taken at the mitotic peak with those in unsynchronized cells.     

Activation of m1 Muscarinic Acetylcholine Receptor Regulates τ Phosphorylation in Transfected PC12 Cells

Abstract: Hyperphosphorylated τ proteins are the principal fibrous component of the neurofibrillary tangle pathology in Alzheimer's disease. The possibility that τ phosphorylation is controlled by cell surface neurotransmitter receptors was examined in PC12 cells transfected with the gene for the rat m₁ muscarinic acetylcholine receptor. Stimulation of m₁ receptor in these cells with two acetylcholine agonists, carbachol and AF102B, decreased τ phosphorylation, as indicated by specific τ monoclonal antibodies that recognize phosphorylation-dependent epitopes and by alkaline phosphatase treatment. The muscarinic effect was both time and dose dependent. In addition, a synergistic effect on τ phosphorylation was found between treatments with muscarinic agonists and nerve growth factor. These studies provide the first evidence for a link between the cholinergic signal transduction system and the neuronal cytoskeleton that can be mediated by regulated phosphorylation of τ microtubule-associated protein.     

Interactions between adaptor protein-1 of the clathrin coat and microtubules via type 1a microtubule-associated proteins.

The classical view suggests that adaptor proteins of the clathrin coat mediate the sorting of cargo protein passengers into clathrin-coated pits and the recruitment of clathrin into budding areas in the donor membrane. In the present study, we provide biochemical and morphological evidence that the adaptor protein 1 (AP-1) adaptor of the trans-Golgi network clathrin interacts with microtubules. AP-1 in cytosolic extracts interacted with in vitro assembled microtubules, and these interactions were inhibited by ATP depletion of the extracts or in the presence of 5'-adenylylimidodiphosphate. An overexpressed gamma-subunit of the AP-1 complex associated with microtubules, suggesting that this subunit may mediate the interaction of AP-1 with the cytoskeleton. Purified AP-1 did not interact with purified microtubules, but interaction occurred when an isolated microtubule-associated protein fraction was added to the reaction mix. The gamma-adaptin subunit of AP-1 specifically co-immunoprecipitated with a microtubule-associated protein of type 1a from rat brain cytosol. This suggests that type 1a microtubule-associated protein may mediate the association of AP-1 with microtubules in the cytoplasm. The microtubule binding activity of AP-1 was markedly inhibited in cytosol of mitotic cells. By means of its interaction with microtubule-associated proteins, we propose novel roles for AP-1 adaptors in modulating the dynamics of the cytoskeleton, the stability and shape of coated organelles, and the loading of nascent AP-1-coated vesicles onto appropriate microtubular tracks.     

Myosin filaments in cytoskeletons of Dictyostelium amoebae

Cytoskeletons were prepared from vegetative amoebae of Dictyostelium discoideum by extraction with Triton X-100. The cytoskeletons were suspended in buffers known to induce the assembly or disassembly of myosin filaments. The samples were fixed, and thin sections were examined by transmission electron microscopy. In both types of buffers, myosin-containing cytoskeletons exhibited a ring of densely staining proteinaceous material within the cortical filament matrix; this ring was not observed in myosin-free cytoskeletons. When myosin-containing cytoskeletons were placed in buffers that induced myosin polymerization, the ring appeared as an array of rodlike filaments approximately 13 nm wide and up to 0.5 micron in length--dimensions appropriate for myosin thick filaments. If ATP was added to cytoskeletons containing such filaments, the cytoskeletons contracted and the ring of filaments disappeared. ATP-induced contraction of cytoskeletons was also visualized by indirect immunofluorescence by using monoclonal antibodies to Dictyostelium myosin. All data were consistent with the identification of the protein ring seen by electron microscopy as cortical myosin. Its location and organization were appropriate for the production of cortical contraction through a sliding filament mechanism.     

Protein interactions with the glucose transporter binding protein GLUT1CBP that provide a link between GLUT1 and the cytoskeleton

Subcellular targeting and the activity of facilitative glucose transporters are likely to be regulated by interactions with cellular proteins. This report describes the identification and characterization of a protein, GLUT1 C-terminal binding protein (GLUT1CBP), that binds via a PDZ domain to the C terminus of GLUT1. The interaction requires the C-terminal four amino acids of GLUT1 and is isoform specific because GLUT1CBP does not interact with the C terminus of GLUT3 or GLUT4. Most rat tissues examined contain both GLUT1CBP and GLUT1 mRNA, whereas only small intestine lacked detectable GLUT1CBP protein. GLUT1CBP is also expressed in primary cultures of neurons and astrocytes, as well as in Chinese hamster ovary, 3T3-L1, Madin-Darby canine kidney, Caco-2, and pheochromocytoma-12 cell lines. GLUT1CBP is able to bind to native GLUT1 extracted from cell membranes, self-associate, or interact with the cytoskeletal proteins myosin VI, alpha-actinin-1, and the kinesin superfamily protein KIF-1B. The presence of a PDZ domain places GLUT1CBP among a growing family of structural and regulatory proteins, many of which are localized to areas of membrane specialization. This and its ability to interact with GLUT1 and cytoskeletal proteins implicate GLUT1CBP in cellular mechanisms for targeting GLUT1 to specific subcellular sites either by tethering the transporter to cytoskeletal motor proteins or by anchoring the transporter to the actin cytoskeleton.     

Renin is sorted to the regulated secretory pathway in transfected PC12 cells by a mechanism which does not require expression of the pro-peptide

The rat pheochromocytoma cell line PC12 targets secretory proteins into two distinct pathways. When DNA encoding human prorenin was transfected into PC12 cells, the protein was sorted into the regulated secretory pathway and released with similar kinetics to noradrenaline upon carbachol stimulation. To determine whether information for targeting prorenin lies within the pro-peptide we have transfected PC12 cells with a construct lacking the pro-peptide coding sequence. The transformed line secretes an apparently fully active enzyme and responds to carbachol stimulation with a rapid release of renin activity. We conclude that the pro-peptide of renin is not essential for targeting the protein to the regulated pathway in PC12 cells.     

Characterization of ATP receptor which mediates norepinephrine release in PC12 cells.

PC12 cells, a rat pheochromocytoma cell line, has been reported to release norepinephrine in response to extracellular ATP in the presence of extracellular Ca2+. The potency order of ATP analogues was adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than adenosine 5'-O-(1-thiotriphosphate) = 2-methylthioadenosine 5'-triphosphate (MeSATP) greater than 2'- and 3'-O-(4-benzoyl-benzoyl)ATP (BzATP) greater than ADP greater than 5-adenylylimidodiphosphate. Adenosine 5'-O-(2-thiodiphosphate), beta, gamma-methyleneadenosine 5'-triphosphate, AMP and adenosine were inactive. The ATP action in the absence of extracellular Ca2+, suggests a small but appreciable contribution of intracellular Ca2+ mobilization, for norepinephrine release. However, for some ATP derivatives, like BzATP, almost no contribution of the phospholipase C-Ca2+ pathway is suggested, based on their low activity in inositol phosphates production. To identify the ATP-receptor protein, PC12 cell membranes were photoaffinity-labeled with [32P]BzATP. SDS-PAGE analysis showed that a 53-kDa protein labeling was inhibited by ATP and its derivatives, as well as by P2-antagonists, suramin and reactive blue 2, which inhibit the nucleotide-induced norepinephrine release. The inhibitory activity of the nucleotides was, in parallel with their potency, to induce norepinephrine release. Despite their inability to release norepinephrine, GTP and GTP gamma S inhibited the BzATP labeling, suggesting the participation of a putative G protein in the ATP-receptor-mediated actions. We suggest that the 53-kDa protein on the PC12 cell surface is an ATP receptor, which mediates the norepinephrine release, depending, mainly, on extracellular Ca2+ gating.