Association of two pertussis toxin-sensitive G-proteins with the D2-dopamine receptor from bovine striatum.

The solubilized D2-dopamine receptor from bovine striatum exhibits high and low affinity states for dopaminergic agonists. Guanine nucleotides and pertussis toxin convert the solubilized receptor from a high affinity state to a low one. A D2-receptor preparation partially purified by affinity chromatography on a haloperidol adsorbent, exhibited agonist-stimulated GTPase activity. [32P]ADP-ribosylation by pertussis toxin of this receptor preparation resulted in the specific labeling of two protein bands corresponding to mol. wts of 39 and 41 kd, in SDS-PAGE. Association of these G-proteins with the receptor was specifically inhibited by Gpp(NH)p. Immunoblot analysis of these G-proteins indicated that the 41- and 39-kd protein bands are analogous to brain Gi and Go respectively. These experiments demonstrate that two distinct pertussis toxin-sensitive G-proteins are functionally associated with bovine striatum D2-dopamine receptor.     

The D2-dopamine receptor of anterior pituitary is functionally associated with a pertussis toxin-sensitive guanine nucleotide binding protein

Dopaminergic inhibition of prolactin release from the anterior pituitary may be mediated through both the adenylate cyclase and Ca2+ mobilization/phosphoinositide pathways. The D2-dopamine receptor of the bovine anterior pituitary has been partially purified by affinity chromatography on CMOS-Sepharose (immobilized carboxymethyleneoximinospiperone). Reinsertion of these partially purified receptor preparations into phospholipid vesicles reconstituted guanine nucleotide-sensitive high affinity agonist binding, agonist-promoted GTPase and 35S-labeled guanosine 5'-O-(thiotriphosphate) [( 35S]GTP gamma S) binding activity in these preparations. Pertussis toxin treatment of the purified receptor preparation abolished agonist-stimulated GTPase and guanine nucleotide-sensitive high affinity agonist binding. These observations suggest that the receptor copurifies with an endogenous, pertussis toxin-sensitive guanine nucleotide binding protein (N). [32P]ADP-ribosylation of affinity-purified D2 receptor preparations by pertussis toxin revealed the presence of a substrate of Mr 39,000-40,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide maps generated using elastase of the [32P]ADP-ribosylated endogenous N protein, transducin, and Ni and No from brain revealed similarities but not identity between the endogenous pituitary N protein and brain Ni and No. Immunoblotting of the partially purified D2 receptor preparations showed an Mr 39,000-40,000 band with an Ni-specific antiserum raised against a synthetic peptide, and with RV3, an No-specific anti-serum, but not with CW6, an antiserum strongly reactive with brain Ni. Several lines of evidence indicate that endogenous pituitary N protein is functionally coupled to the D2 receptor. As measured by [35S]GTP gamma S binding, ratios of 0.2-0.6 mol N protein/mol receptor were observed. Association of N protein with the D2 receptor was increased by agonist pretreatment and decreased by guanine nucleotides. These results suggest that No and/or a form of Ni distinct from the Mr 41,000 pertussis toxin substrate (Ni) is the predominant N protein functionally coupled with the D2-dopamine receptor of anterior pituitary.     

Evidence that human platelet alpha-adrenergic receptors coupled to inhibition of adenylate cyclase are not associated with the subunit of adenylate cyclase ADP-ribosylated by cholera toxin

Exposure of the alpha-adrenergic receptor of the human platelet to agonist prior to solubilization stabilizes a receptor complex of the alpha-adrenergic receptor with the GTP-binding protein(s) which modulates receptor affinity for agonists (Smith, S. K., and Limbird, L. E. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4026-4030). The soluble alpha-adrenergic receptor is characterized by retention of sensitivity to GTP and a faster rate of sedimentation in sucrose gradients than antagonist-occupied or unoccupied receptors. The present studies were undertaken to determine whether the alpha-adrenergic receptor, which is coupled to inhibition of adenylate cyclase, contains the same GTP-binding protein that is involved in activation of adenylate cyclase. The GTP-binding protein that is coupled to activation of adenylate cyclase was labeled with [32P]ADP-ribose using cholera toxin. Incorporation of [32]ADP-ribose into a Mr = 42,000 peptide in human platelet membranes was paralleled by an enhancement of GTP-sensitive catalytic activity in the membranes. However, cholera toxin treatment did not modify alpha-receptor-mediated inhibition of adenylate cyclase or interaction of the alpha-receptor with agonist agents. Moreover, sucrose gradient centrifugation revealed that the [32P]ADP-ribosylated Mr = 42,000 subunit of the stimulatory GTP-binding protein did not appear to associate with the agonist-alpha-receptor complex. These data suggest that the GTP-binding protein that mediates GTP activation of adenylate cyclase in the human platelet membrane is distinct from the GTP-binding protein that modulates alpha-adrenergic receptor affinity for agonist agents and which associates with the receptor in the presence of agonists.     

The magnetic properties of the nickel cofactor F430 in the enzyme methyl-coenzyme M reductase of Methanobacterium thermoautotrophicum.

Preparations of gamma-aminobutyrate (GABA)/benzodiazepine receptor from pig cerebral cortex are composed of three major bands of polypeptides (51, 55 and 57 kDa) which are purified in a ratio of approx. 2:1:1 respectively. Treatment of purified receptor preparations with cyclic AMP-dependent protein kinase resulted in major incorporation of 32P into the 55 kDa band only. The maximum incorporation achieved was 0.6 mol of 32P/mol of 55 kDa polypeptide. The phosphorylated receptor subunit (beta-subunit) displays the same apparent Mr as a band labelled irreversibly with the GABA receptor agonist [3H]muscimol. The two nonphosphorylated subunit polypeptides (51 and 57 kDa) are each labelled irreversibly with [3H]flunitrazepam and are recognized by anti-peptide antibodies specific for alpha-subunits.     

Identification of the insulin receptor tyrosine residues undergoing insulin-stimulated phosphorylation in intact rat hepatoma cells

Tyr(P)-containing proteins were purified from extracts of insulin-treated rat hepatoma cells (H4-II-E-C3) by antiphosphotyrosine immunoaffinity chromatography. Two major insulin-stimulated, Tyr(P) proteins were recovered: an Mr 95,000 protein (identified as the insulin receptor beta subunit by its immunoprecipitation by a patient-derived anti-insulin receptor serum and several anti-insulin receptor (peptide) antisera) and an Mr 180,000 protein (which was unreactive with all anti-insulin receptor antibodies). After purification and tryptic digestion of the Mr 95,000 protein, tryptic peptides containing Tyr(P) were purified by sequential antiphosphotyrosine immunoaffinity, reversed-phase, anion-exchange chromatography. The partial amino acid sequence obtained by gas- and solid-phase Edman degradation was compared to the amino acid sequence of the intracellular extension of the rat insulin receptor deduced from the genomic sequence. Approximately 80% of all beta subunit [32P]Tyr(P) resides on two tryptic peptides: 50-60% of [32P]Tyr(P) is found on the tryptic peptide Asp-Ile-Tyr-Glu-Thr-Asp-Tyr-Tyr-Arg from the tyrosine kinase domain, which is recovered mainly as the double phosphorylated species (predominantly in the form with Tyr(P) at residues 3 and 7 from the amino terminus; the remainder with Tyr(P) at residues 3 and 8), with 10-15% as the triple phosphorylated species. A second tryptic peptide is located near the carboxyl terminus, contains 2 tyrosines, and has the sequence, Thr-Tyr-Asp-Glu-His-Ile-Pro-Tyr-Thr-; this contains 20-30% of beta subunit [32P]Tyr(P) and is identified primarily in a double phosphorylated form. Approximately 10% of beta subunit [32P]Tyr(P) resides on an unidentified tryptic peptide of Mr 4,000-5,000. The insulin-stimulated tyrosine phosphorylation of the insulin receptor in intact rat hepatoma cells thus involves at least 6 of the 13 tyrosine residues located on the beta subunit intracellular extension. These tyrosines are clustered in several domains in a distribution virtually identical to that previously found for partially purified human insulin receptor autophosphorylated in vitro in the presence of insulin. This multisite regulatory tyrosine phosphorylation is the initial intracellular event in insulin action.     

The beta subunit of the insulin receptor is an insulin-activated protein kinase

In the presence of adenosine 5'-[gamma-32P]triphosphate ([gamma-32P]ATP) and a partially purified human placental insulin receptor preparation, insulin stimulates the phosphorylation of an Mr 94000 protein in a time- and dose-dependent manner. Half-maximal stimulation of 32P incorporation occurs at (2-3) X 10(-9) M insulin, a concentration identical with the Kd for insulin binding in this preparation. Immunoprecipitations with monoclonal anti-insulin receptor antibody demonstrate that the Mr 94000 protein kinase substrate is a component of the insulin receptor, the beta subunit. If the partially purified, soluble placental receptor preparation is immunoprecipitated and then exposed to [gamma-32P]ATP and insulin, phosphorylation of the Mr 94000 protein is maintained. The photoincorporation of 8-azido[alpha-32P]ATP into placental insulin receptor preparations was carried out to identify the ATP binding site responsible for the protein kinase activity. Photoincorporation into numerous proteins was observed, including both subunits of the insulin receptor. However, when photolabeling was performed in the presence of excess adenosine 5'-(beta, gamma-imidotriphosphate), a nonhydrolyzable ATP derivative, the beta subunit of the insulin receptor was the only species protected from label incorporation. These data indicate that the beta subunit of the insulin receptor has insulin-dependent protein kinase activity. Phosphotyrosine formation is the primary result of this activity in placental insulin receptor preparations.     

Polymorphic forms of human apolipoprotein[a]: inheritance and relationship of their molecular weights to plasma levels of lipoprotein[a]

The plasma concentration of human lipoprotein[a], Lp[a], is highly correlated with coronary artery disease. The protein moiety of Lp[a], apoLp[a], consists of two apoproteins, apo[a] and apoB-100, linked by one or more disulfide bonds(s). Apo[a], the protein unique to Lp[a], exists in polymorphic forms that exhibit different apparent molecular weights (Mr). Polyacrylamide gel electrophoresis in sodium dodecyl sulfate followed by immunoblotting was used to separate and visualize these different forms and to determine the polymorphic pattern of apo[a] in the plasma samples of 692 individuals. A total of 11 different polymorph bands ranging in Mr from 419 kD to 838 kD could be resolved, but only 1 or 2 bands were present per individual. The polymorphic band pattern for an individual was assigned to 1 of the 66 different phenotype designations representing the total number of possible single- and double-band combinations of the 11 detectable bands. All 11 of the possible single-band phenotypes but only 32 of the 55 possible double-band phenotypes were represented. There were 412 plasma samples (59.5%) that contained a single band, 274 (39.6%) contained two bands, and only 6 (0.9%) had no detectable apo[a] band. A highly significant inverse correlation was found between the Mr of the band(s) present and the plasma apoLp[a] concentration (r = -0.461; rho = 0.0001). The correlation was better between apoLp[a] and single-band (r = -0.495; rho = 0.0001) than double-band (r = -0.382; rho = 0.0001) phenotypes. Of the 274 individuals exhibiting double-band phenotypes, the lower Mr band was more intense in 141 (51.4%), the two bands were equally intense in 85 (31.0%), while the higher Mr band was more intense in 48 (17.5%). Based upon the hypothesis that apo[a] polymorphism is controlled by different alleles at a single locus, the frequency of the 11 alleles determined from the observe phenotypes (low Mr----high Mr) was: band 1) 419 kD, 0.00875; band 2) 489 kD, 0.00510; band 3) 536 kD, 0.0555; band 4) 553 kD, 0.0758; band 5) 613 kD, 0.135; band 6) 680 kD, 0.0824; band 7) 705 kD, 0.104; band 8) 742 kD, 0.151; band 9) 760 kD, 0.246; band 10) 796 kD, 0.128; band 11) 838 kD, 0.00802. The observed distribution of phenotypes in the population was compared by chi-square analysis to that predicted on the basis of simple Mendelian inheritance, and the hypothesis was rejected (chi 2 = 921.7; rho less than 0.001). Significantly, the singleband phenotypes are over-represented in the population compared to that predicted.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chemical and functional analysis of components of adenylyl cyclase from human platelets treated with phorbolesters

Human platelets, prelabeled with [32P]phosphate were treated with tetradecanoylphorbol acetate (TPA) for 5 min at 37 degrees C. Phosphorylation of the components of adenylyl cyclase was determined in membranes using specific antibodies against G-proteins and the catalytic moiety. Less than 0.01 mol of [32P]phosphate/mol could be detected in immunoprecipitates using antibodies against sequences within the alpha-subunit of the GTP binding protein Gi. TPA, however, caused the incorporation of 0.67-1.1 mol of [32P]phosphate per mol of catalyst while 0.13-0.2 mol were found in the absence of TPA. Lack of modification of the alpha-subunit of Gi was also indicated by the results of reconstitution experiments with purified Gi alpha from bovine brain: adenylyl cyclase in membranes from untreated platelets was significantly more inhibited by added G1 alpha, than that from TPA treated cells. While beta, gamma-subunits were like-wise inhibitory no difference dependent on platelet-pretreatment could be observed.     

Ligand-induced formation of the leukotriene B4 receptor-G protein complex of human polymorphonuclear leukocytes.

The components of the polymorphonuclear leukocyte (PMNL) receptor for leukotriene B4 (LTB4) were examined by Sephacryl S-300 exclusion chromatography of PMNL membrane proteins, which were solubilized before and after the binding of [3H] LTB4. When the PMNL membranes were solubilized in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and filtered on Sephacryl S-300 prior to addition of [3H] LTB4, the binding activity was associated with a 65 kD protein. In contrast, the radioactivity of [3H] LTB4 bound to PMNL membranes prior to solubilization was recovered predominantly with a 140 kD protein. When PMNL membranes had been pretreated with pertussis toxin, but not cholera toxin, before the addition of LTB4 and subsequent solubilization, radioactivity was recovered predominantly with the 65 kD protein. The addition of guanylylimidodiphosphate (GMP-PNP), a nonhydrolyzable derivative of guanosine triphosphate (GTP), to PMNL membrane receptors bearing [3H] LTB4 either prior to or after CHAPS solubilization reduced the yield of the 140 kD presumed LTB4 receptor protein-G protein complex. That the maximum specific binding of [35S] guanosine-5'-0-3-thiotriphosphate (GTP-gammaS) to LTB4-binding proteins in the Sephacryl S-300 effluent corresponded to the 140 kD protein supported the presence of a G protein in the LTB4 receptor complex.     

Identification of proteins resembling G-protein alpha subunits in locust muscle

In locust skeletal muscle, FMRFamide-like peptides decrease a K+ conductance. Functional data suggest the involvement of G-proteins. For identification of G-protein alpha-subunits, membranes of locust skeletal muscle were probed with ADP-ribosylating bacterial toxins, the photoreactive GTP analog, [alpha-32P]GTP azidoanilide, and with antibodies against mammalian alpha-subunits. Multiple guanine nucleotide-binding proteins of approximately 24-95 kDa were detected. Pertussis toxin catalyzed the ADP-ribosylation of two proteins comigrating with the ADP-ribosylated alpha-subunits of the mammalian G-proteins Go and Gi. Cholera toxin promoted ADP-ribosylation of a protein comigrating with mammalian cholera toxin substrates (i.e., Gs alpha-subunits). An antibody against mammalian Go alpha-subunits detected a 54-kDa protein. Thus proteins with properties of mammalian G-protein subunits are present in insect muscle.     

Phosphorylation of rat liver glucocorticoid receptor

Rat liver glucocorticoid-receptor complex (GRc) was purified 2000-fold by a combination of methods including (NH4)2SO4-fractionation and phosphocellulose and DNA-cellulose chromatography. The purified glucocorticoid receptor preparation contained a major peptide of Mr = 90,000 and the GRc sedimented as 4 S in 5-20% sucrose gradients. An additional peptide of Mr = 45,000 (45K) was also observed. Some preparations yielded only the Mr = 90,000 (90K) peptide suggesting that the 45K peptide may be a proteolyzed portion of the 90K protein. The purified GRc was incubated with [gamma-32P]ATP in the presence of cAMP-dependent kinase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the above preparation revealed the presence of two 32P-containing bands with apparent Mr = 90,000 and 45,000. The 32P incorporation was dependent on the availability of divalent cation (Mg2+). GRc in cytosol labeled with [3H]dexamethasone mesylate and purified as above co-migrated with 32P-containing bands. GRc was also purified from cytosol obtained from livers of rats injected with [32P]orthophosphate. Both 32P and 3H bands were associated with 90K and 45K peptides. Our results indicate that rat liver glucocorticoid receptor is a phosphoprotein and that both the phosphorylated peptides 90K and 45K also contain the steroid and the DNA binding regions of the glucocorticoid receptor.     

Autophosphorylation and rapid dephosphorylation of the cAMP-dependent protein kinase from Blastocladiella emersonii zoospores

The photoaffinity label 8-azido[32P]adenosine 3':5'-monophosphate and affinity chromatography on N6-(2-aminoethyl)-cAMP-Sepharose were used to analyze the cAMP-binding proteins present in cell-free extracts of Blastocladiella emersonii zoospores. In the presence of a mixture of protease inhibitors, 8-azido[32P]cAMP was specifically and quantitatively incorporated into a major protein band of Mr = 58,000, and three minor protein bands of Mr = 50,000, Mr = 43,000, and Mr = 36,000 respectively, after autoradiography following sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. In the absence of the protease inhibitors, the Mr = 58,000 protein band was converted into the lower molecular weight cAMP-binding proteins, indicating a high sensitivity of the intact Mr = 58,000 protein band to endogenous proteases. The Mr = 58,000 protein corresponded to the regulatory subunit (R), of the cAMP-dependent protein kinase of zoospores, as shown by their identical behavior on DEAE-cellulose chromatography. The partially purified protein kinase incorporated 32P from [gamma-32P] ATP . Mg2+ into R as demonstrated by the specific adsorption of the 32P-labeled protein with N6-(2-aminoethyl)-cAMP-Sepharose. The incorporated 32P group was rapidly removed by endogenous phosphoprotein phosphatases in the presence of cAMP, as shown by pulse-chase experiments with [gamma-32P]ATP. Dephosphorylation of R-cAMP and rapid proteolysis may indicate two other mechanisms, in addition to cAMP, for the control of this protein kinase in vivo.     

4-Azido-2-nitrophenyl phosphate, a new photoaffinity derivative of inorganic phosphate. Study of its interaction with the inorganic phosphate binding site of beef heart mitochondrial adenosine triphosphatase

4-Azido-2-nitrophenyl phosphate (ANPP) was synthesized and characterized. ANPP, unlabeled or labeled by 32P, was used as a photoreactive analogue of Pi to study the Pi binding site(s) in isolated F1-ATPase and inside-out particles from beef heart mitochondria. In the dark, the phosphate bond of ANPP was cleaved by alkaline phosphatase but not by mitochondrial F1-ATPase. ANPP bound reversibly to the phosphate site of F1-ATPase as shown by competitive inhibition of binding of Pi to F1-ATPase by ANPP in the dark; the Ki value was 60 microM. Upon photoirradiation with visible light, [32P]ANPP bound covalently to F1-ATPase and inactivated the enzyme. Part of the added ANPP was, however, photolyzed with release of Pi. By extrapolation, it could be calculated that complete inactivatin of F1-ATPase was accompanied by incorporation of 32P radioactivity corresponding to 1 mol of [32P]ANPP per mol of F1-ATPase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [32P]-ANPP-labeled F1-ATPase revealed only one radioactive peptide with a Mr of 50000. This peptide was characterized as the beta subunit of F1-ATPase by specific labeling with [14C]dicyclohexylcarbodiimide [Pougeois, R., Satre, M., & Vignais, P. V. (1979) Biochemistry 18, 1408-1413]. Photoirradiation of inside-out submitochondrial particles with [32P]ANPP resulted in the labeling of two peptides with a Mr of 50000 and 30000-32000; both labelings were significantly decreased by incubation of the particles with Pi prior to photoirradiation. The Mr 50000 peptide is most probably the beta subunit of F1-ATPase; the other peptide might be the Pi carrier protein.     

Characterization of the human melanoma nerve growth factor receptor

Monoclonal antibodies to the human nerve growth factor receptor have been used to biochemically characterize the receptor in the human melanoma cell line A875. Labeling of A875 cell proteins by culture with [35S]cysteine or labeling of cell surface proteins with 125I followed by immunoprecipitation with anti-nerve growth factor receptor antibody reveals a receptor protein with an apparent Mr of 70,000-75,000 and an isoelectric point of 4.9-5.2. Incorporation of [3H]glucosamine into this species indicates it is a glycoprotein. The receptor becomes phosphorylated on serine residues in intact cells and in isolated membranes incubated with [gamma-32P]ATP. The receptor appears to exist, at least partially, in the form of a disulfide-linked oligomer (probably a dimer) of Mr = 75,000 subunits. Kinetic [35S]cysteine labeling studies reveal an Mr = 59,000 core protein which is glycosylated via N-linked and probably also O-linked sugar moieties to produce the mature (Mr = 70,000-75,000) receptor.     

Agonist-dependent, cholera toxin-catalyzed ADP-ribosylation of pertussis toxin-sensitive G-proteins following transfection of the human alpha 2-C10 adrenergic receptor into rat 1 fibroblasts. Evidence for the direct interaction of a single receptor with two pertussis toxin-sensitive G-proteins, Gi2 and Gi3.

A DNA encoding the human alpha 2-C10 adrenergic receptor was transfected into Rat 1 fibroblasts and clones selected on the basis of resistance to G418 sulfate. Two clones, one of which (1C) expressed some 3.5 pmol/mg membrane protein of the receptor as assessed by the specific binding of [3H]yohimbine and one (4D) which did not express detectable amounts of the receptor were selected for further study. When cholera toxin-catalyzed ADP-ribosylation was performed with [32P]NAD on membranes of these cells in the absence of added guanine nucleotides, radioactivity was incorporated into a polypeptide(s) of 40 kDa in addition to the 45- and 42-kDa forms of Gs alpha. Addition of the selective alpha 2 receptor agonist U.K.14304 enhanced markedly, in a dose-dependent manner, the cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s), but not the 45- or 42-kDa polypeptides, in membranes of the 1C cells. Dose response curves for U.K.14304 enhancement of cholera toxin-labeling of the 40-kDa polypeptide(s) and stimulation of high affinity GTPase activity were identical. By contrast, U.K.14304 was ineffective in either assay in membranes from the 4D cells, demonstrating this effect to be dependent upon receptor activation. Furthermore, the alpha 2 receptor antagonist yohimbine blocked all effects of U.K.14304. The agonist promotion of cholera toxin-catalyzed ADP-ribosylation of Gi was completely blocked by guanine nucleotides. Whether GDP or GDP + fluoroaluminate (as a mimic of GTP) was used, blockade of the agonist effect was complete and indeed both conditions prevented agonist-independent labeling by cholera toxin of the 40-kDa polypeptide(s). Mg2+ produced an agonist-independent cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s) but even in the presence of [Mg2+], agonist-stimulation of cholera toxin-labeling of the 40-kDa polypeptide(s) was observed and was additive with the effect of [Mg2+]. Agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi was completely attenuated by pretreatment of the cells with pertussis toxin, which prevents contact between receptors and G-proteins which are substrates for this toxin. By contrast, pretreatment of the cells with concentrations of cholera toxin able to "down-regulate" essentially all of the membrane-associated Gs alpha did not prevent agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sequence of prolactin effects on phospholipid synthesis in Nb2 node lymphoma cells

In Nb2 node rat lymphoma cells, the effects of prolactin (PRL) on the rates of incorporation of several precursors into neutral lipids, phospholipids and proteins were determined. The onset of the PRL stimulation of radiolabeled-precursor incorporation into lipids occurred between 1 and 4 hours after PRL addition to Nb2 cells; precursors employed included [14C]-acetate, [3H]-glycerol, [32P]O4, [3H]-choline, [3H]-ethanolamine, [3H]-serine and [3H]-myoinositol. No effects were observed during the initial 60 min of culture with PRL. The effects on precursor incorporation that occur after 1 hr of PRL exposure are likely related to the stimulation of cell growth by PRL. In cells that were prelabeled with the radiolabeled precursors and subsequently incubated with PRL, PRL had no effect on the metabolism of the radiolabeled phospholipids or the accumulation of phospholipid products until several hours after hormone addition. We would conclude from these studies that the initial (60 min) effect of PRL on Nb2 node lymphoma cells does not likely use a signal transduction mechanism that involves products derived from the cellular phospholipids.     

Prolactin receptor triggering. Evidence for rapid tyrosine kinase activation.

The mechanism of action of prolactin (PRL) has remained obscure despite the unveiling of the primary structure of PRL receptors. The present study demonstrates rapid PRL receptor-mediated tyrosine phosphorylation of at least three cellular proteins, designated p120, p97, and p40, in a rat T-lymphoma (Nb2-11C) as revealed by antiphosphotyrosine immunoblotting. One of the phosphotyrosyl proteins, p120, co-purified with activated PRL receptor complexes obtained using either anti-ligand or anti-receptor antibodies. Furthermore, in vitro incubation of affinity-purified PRL receptor complexes from PRL-stimulated cells with ATP in the presence of a tyrosine phosphatase inhibitor, resulted in a 10-15-fold increase in the phosphotyrosine content of p120, as revealed by antiphosphotyrosine immunoblotting. Parallel experiments utilizing [gamma-32P]ATP confirmed a rapid and time-dependent incorporation of phosphate into p120 in the same affinity-purified PRL receptor complexes. These data provide strong evidence for the involvement of a tyrosine kinase in PRL signal transduction and suggest the presence of a tyrosine kinase within the activated PRL receptor complex.     

The surface cyclic AMP receptor in Dictyostelium. Levels of ligand-induced phosphorylation, solubilization, identification of primary transcript, and developmental regulation of expression

A monospecific polyclonal antiserum to the surface cAMP receptor of Dictyostelium has been developed by immunization with purified receptor immobilized on particles of polyacrylamide and on nitrocellulose paper. In Western blots, the antiserum displays high affinity and specificity for both the R (Mr 40,000) and D (Mr 43,000) forms of the receptor previously identified by photoaffinity labeling with 8-azido-[32P] cAMP. These bands, labeled with the photoaffinity label or with 32 Pi, were quantitatively and specifically immunoprecipitated, supporting co-purification data that all represent the same polypeptide. The R form, found in unstimulated cells, contained at least 0.2 mol of phosphate/mol of receptor. The D form, generated by cAMP stimulation of intact cells, contained at least 4 mol of phosphate/mol of receptor. In the absence of detergents, the receptor was exclusively located on membranes. The receptor was solubilized effectively in Triton X-100 and sedimented as a broad peak of 5-7 S on sucrose velocity gradients. Western blots of membranes isolated at different times after starvation indicate that the appearance of cell surface cAMP binding sites during the aggregation stage of development (5-6 h) is due to de novo synthesis of receptor protein. Pulse labeling with [35S]methionine indicated that the receptor is most rapidly synthesized during the preaggregation stage of development (1-3 h), prior to its maximal accumulation in membranes. The serum specifically immunoprecipitates a polypeptide of Mr 37,000 from an in vitro translation reaction using RNA isolated from preaggregation stage cells. The time course of expression of the mRNA coding for the Mr 37,000 polypeptide parallels the rate of receptor synthesis in vivo.     

Evidence for receptor-mediated inhibition of intrinsic activity of GTP-binding protein, Gi1 and Gi2, but not G0 in reconstitution experiments

The receptor-mediated inhibition of intrinsic activities of GTP-binding proteins (G-proteins) was studied. Pertussis toxin (IAP)-substrate G-protein, Gi1, Gi2 or G0, was prelabeled with [alpha-32P]GDP and reconstituted with synaptic membranes of the guinea pig cerebellum in the presence of 0.02% of Chaps. Intrinsic activities of G-proteins were evaluated by the release of [alpha-32P]GDP in exchange for added GppNHp or GDP in reconstituted preparations. U-50,488H (1 nM-10 microM), a specific kappa-subtype of opioid receptor agonist, inhibited the [alpha-32P]GDP release in exchange for added 1 microM GppNHp in Gi1-reconstituted preparations in a concentration-dependent manner. On the other hand, the kappa-opioid agonist at 10 microM increases the Km values of GppNHp, but not GDP in exchange for [alpha-32P]GDP release in preparations reconstituted with Gi1 or Gi2, but not with G0. These findings indicate that kappa-opioid receptor is coupled to inhibition of intrinsic activities of Gi1 and Gi2, but not G0, in guinea pig cerebellar membranes. In addition, it was revealed that the mode of action is mediated by a decrease in affinity of GTP (or its analog) for G proteins, but not by a change in affinity of GDP.     

Rat heart cell membranes contain three substrates for cholera toxin-catalyzed ADP-ribosylation and a single substrate for pertussis toxin-catalyzed ADP-ribosylation

Three peptides (Mr = 45,000, 47,000 and 52,000) in the cholate extract from rat heart cell membranes were radiolabeled when the extract was incubated in the presence of activated cholera toxin and [32P]NAD. A single peptide of Mr = 41,000 in this extract was ADP-ribosylated by pertussis toxin in the presence of [32P]NAD.