Covalent cross-linking of prostaglandin E receptor from bovine adrenal medulla with a pertussis toxin-insensitive guanine nucleotide-binding protein

Prostaglandin E2 (PGE2) specifically bound to 100,000 X g pellet prepared from bovine adrenal medulla, and [3H]PGE2-bound proteins were solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The dissociation of bound [3H]PGE2 from the proteins was enhanced by GTP. [3H]PGE2-specifically bound proteins were adsorbed onto a wheat germ agglutinin column and GTP treatment decreased the amount of [3H]PGE2 retained on the column. When [3H]PGE2-bound proteins were cross-linked in the membrane by dithiobis(succinimidyl propionate) and solubilized, bound [3H]PGE2 was no longer dissociated by GTP treatment, suggesting that cross-linking produced a stable and high-affinity complex of PGE receptor with a GTP-binding protein. Covalent cross-linking of the complex was attested by adsorption of dithiobis(succinimidyl propionate)-treated [3H]PGE2-bound proteins to GTP-Sepharose, and co-elution of [35S]guanosine 5'-O-(3-thiotriphosphate) binding activity and immunoreactivities of alpha o and beta subunits of a GTP-binding protein. The cross-linked [3H]PGE2-bound complex was eluted as an apparently single radioactive peak at the position of Mr = 200,000 by gel filtration. These results have demonstrated that PGE receptor is a glycoprotein with an approximate Mr of 110,000, assuming that the Mr of the GTP-binding protein is 90,000. PGE2 neither activated nor inhibited adenylate cyclase activity, and pertussis toxin (islet-activating protein) did not affect PGE2 binding and its GTP sensitivity. These results suggest that the PGE receptor may be functionally associated with a pertussis toxin-insensitive GTP-binding protein and is not coupled to the adenylate cyclase system in bovine adrenal medulla.     

Identification of the prostacyclin receptor by use of [15-3H1]19-(3-azidophenyl)-20-norisocarbacyclin, an irreversible specific photoaffinity probe.

The prostaglandin I (PGI2) receptor of mouse mastocytoma P-815 cells was characterized by photo-affinity labeling with the stable PGI2 analogue [15-3H1]-19-(3-azidophenyl)-20-norisocarbacyclin ([3H] APNIC) used as a potential photoaffinity probe for the receptor. [3H]APNIC bound to the mastocytoma membrane with high affinity and in a saturable manner. Scatchard plot analysis indicated a single binding site with a Kd of 4.7 nM and a Bmax of 0.58 pmol/mg protein. The binding of [3H]APNIC was dose dependently inhibited by APNIC and iloprost, another stable PGI2 agonist, and to a much lesser extent by PGE2. The binding of the radioligand showed sensitivity to the guanine nucleotide guanosine 5'-O-(3-thio-triphosphate) (GTP gamma S). Photolysis of [3H]APNIC-prelabeled membranes resulted in incorporation of radiolabel into a protein of approximately 43 kDa. Photolabeling was inhibited by PGI2 agonists and other prostaglandins with specificity for the PGI2 receptor and was modulated by GTP gamma S. A protein of approximately 45 kDa was also labeled by [3H]APNIC in the membrane of porcine platelets, membranes that are known to be abundant in PGI2 receptors. These results demonstrate that [3H]APNIC specifically labels a protein that may represent the PGI2 receptor and that this radioprobe will be a useful reagent for further characterization and purification of the PGI2 receptor.     

Solubilization of rat liver vasopressin receptors as a complex with a guanine-nucleotide-binding protein and phosphoinositide-specific phospholipase C.

Vasopressin V1 receptors were solubilized from rat liver plasma membranes with the detergent lysophosphatidylcholine. [[3H]Arginine]vasopressin (AVP) binding to the solubilized preparations was specific and saturable, with a dissociation constant of 0.6 nM. Cross-linking of [125I]vasopressin to the solubilized fraction, studied by SDS/polyacrylamide-gel-electrophoretic analysis, demonstrated the presence of a 65 kDa band which was specifically labelled with [125I]vasopressin. Specific binding of [3H]AVP to these solubilized receptors was decreased by guanine nucleotides, but not by adenosine 5'-[beta gamma-imido]triphosphate. Addition of vasopressin increased specific binding of 35S-labelled guanosine 5'-[gamma-thio]triphosphate (GTP[35S]) to the solubilized fractions, indicating co-solubilization of GTP-binding protein(s) [G-protein(s)] and vasopressin receptors. The solubilized fraction was insensitive to both cholera- and pertussistoxin treatment. Immunoblotting of the solubilized fraction with antibodies specific for a phosphoinositide-specific phospholipase C (PI-PLC I) demonstrated the presence of a 60 kDa protein. Anti-PI-PLC I antiserum immunoprecipitated solubilized vasopressin-binding sites from rat liver (V1), but not solubilized vasopressin-binding sites from hog kidney (V2). Similar results were obtained with an anti-PI-PLC I IgG affinity column. The solubilized (V1) receptors were enriched by ion-exchange and high-performance gel-filtration liquid chromatography. Vasopressin-binding activity was co-eluted with PI-PLC I and GTP[S]-binding activity on a DEAE-Sepharose column. The major vasopressin- and GTP[35S]-binding activities were co-eluted with PI-PLC I activity at approx. 240 kDa suggesting that vasopressin receptors from rat liver membranes can be solubilized as a complex of receptor-coupler-effector by using the detergent lysophosphatidycholine.     

Characterization of the guinea pig lung membrane leukotriene D4 receptor solubilized in an active form. Association and dissociation with an islet-activating protein-sensitive guanine nucleotide-binding protein

Membrane fractions from the guinea pig lung had high- and low-affinity binding sites for LTD4 with Kd values of 0.016 and 9.1 nM, respectively. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or by prior treatment of the membrane with islet-activating protein (IAP), the high-affinity site shifted to a low-affinity state. Consistently, a 41-kDa protein was ADP-ribosylated by treatment of the lung membranes with IAP, and this event was inhibited by the addition of GTP gamma S. We solubilized the LTD4 receptor from the lung membranes in an active form with 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 10% glycerol. On a gel filtration column, the binding activity was eluted at the volume corresponding to a Mr of 70,000 or over 500,000 in the presence or absence of Mg2+ (5-20 mM), respectively, in solubilizing buffers. The Kd value of [3H]LTD4 binding to the 70-kDa protein was similar to the low-affinity binding constant of the membrane and was insensitive to GTP gamma S. The preparation solubilized in the absence of Mg2+ showed both high- and low-affinity binding sites for LTD4, and the addition of GTP gamma S shifted the high-affinity site to a low-affinity one. Thus, 1) the LTD4 receptor is coupled to an IAP-sensitive GTP-binding protein, 2) this GTP-binding protein is dissociable from the receptor by solubilizing the lung membrane with CHAPS and Mg2+, and 3) the receptor associated to or dissociated from a GTP-binding protein exhibited a high- or low-affinity state, respectively. These data provide an insight into the molecular mechanism of regulation of the LTD4 receptor signaling process by association and dissociation with an IAP-sensitive GTP-binding protein.     

Solubilization of receptors for thyrotropin-releasing hormone from GH4C1 rat pituitary cells: demonstration of guanyl nucleotide sensitivity

TRH receptors have been solubilized from GH4C1 cells using the plant glycoside digitonin. Solubilized receptors retain the principal binding characteristics exhibited by the TRH receptor in intact pituitary cells and their membranes. The binding of the methylhistidyl derivative of TRH [( 3H]MeTRH) attained equilibrium within 2-3 h at 4 C, and it was reversible, dissociating with a t1/2 of 7 h. Analysis of [3H]MeTRH binding to soluble receptors at 4 C yielded a dissociation constant (Kd) of 3.8 nM and a total binding capacity (Bmax) of 3.9 pmol/mg protein. Peptides known to interact with non-TRH receptors on GH cells failed to interfere with the binding of [3H]MeTRH, indicating that the TRH binding was specific. Chlordiazepoxide, a competitive antagonist for TRH action in GH cells, inhibited TRH binding to soluble receptors with an IC50 of 11 microM. When [3H]MeTRH was bound to membranes and the membrane proteins were then solubilized, we found enhanced dissociation of the prebound [3H]MeTRH from its solubilized receptor by guanyl nucleotides. Maximal enhancement of [3H]MeTRH dissociation by 10 microM GTP gamma S occurred within about 45 min at 22 C. GTP gamma S, GTP, GDP beta S, and GDP were all effectors of [3H]MeTRH dissociation, exhibiting EC50s in the range of 14-450 nM. The rank order of potency of the tested nucleotides was GTP gamma S greater than GTP congruent to GDP beta S greater than GDP much greater than ATP gamma S greater than GMP. We conclude that TRH receptors have been solubilized from GH cells with digitonin and retain the binding characteristics of TRH receptors in intact pituitary cells. Furthermore, prebinding [3H]MeTRH to GH4C1 cell membranes results in the solubilization of a complex in which the TRH receptor is linked functionally to a GTP binding protein.     

Characterization of cytosolic pertussis toxin-sensitive GTP-binding protein in mastocytoma P-815 cells

We have characterized a soluble pertussis toxin (PT)-sensitive GTP-binding protein (G-protein) present in mouse mastocytoma P-815 cells. 65% of total ADP-ribosylation of PT substrate having a molecular mass of 40 kDa on SDS-polyacrylamide gel electrophoresis in cell homogenate was detected in the supernatant after centrifugation at 100,000 x g for 90 min. [32P]ADP-ribosylation of cytosolic PT substrate was significantly enhanced on the addition of exogenous beta gamma complex. The molecular mass of the cytosolic PT substrate was estimated to be about 80 kDa on an Ultrogel AcA 44 column, but the beta gamma complex was not detected in the cytosol by using the anti-beta gamma complex antibody. Furthermore, the cytosolic PT substrate was found to have some unique properties: [35S]GTP gamma S binding was not inhibited by GDP and [32P]ADP-ribosylation was not affected by GTP gamma S treatment. Only after the cytosolic PT substrate had been mixed with exogenous beta gamma complex, did it copurify with exogenous beta gamma complex by several column chromatographies including an Octyl-Sepharose CL-4B column. The PT substrate was identified as Gi2 alpha by Western blot analysis and peptide mapping with S. aureus V8 protease. These results suggest that Gi2 alpha without beta gamma complex exists with an apparent molecular mass of about 80 kDa in the cytosolic fraction of P-815 cells.     

Activation of signal-transducing guanine-nucleotide-binding regulatory proteins by guanosine 5'-[gamma-thio]triphosphate. Information transfer by intermediately thiophosphorylated beta gamma subunits

Signal-transducing guanine-nucleotide-binding regulatory proteins (G proteins) are heterotrimers, composed of the nucleotide-binding alpha subunit and a beta gamma dimer. The influence of beta gamma dimer preparations of the retinal G protein transducin (TD) was studied on formylpeptide-receptor--G-protein interactions in membranes of differentiated HL 60 cells. For this, TD was prepared from bovine rod outer segment (ROS) membranes with either GTP or its analogs, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imino]triphosphate (Gpp[NH]p). After removal of free nucleotides, TD beta gamma was separated from TD alpha and its function analyzed. Addition of TD beta gamma isolated from TD prepared with GTP[S] (TD beta gamma GTP[S]) to HL 60 membranes abolished high-affinity binding of fMet-Leu-[3H]Phe (fMet, N-formylmethionine) to its receptor. In contrast, TD beta gamma isolated from TD prepared with GTP (TD beta gamma GTP), boiled TD beta gamma GTP[S] and TD alpha prepared with GTP[S] had no or only slight effects. The inhibitory effect of TD beta gamma GTP[S] on fMet-Leu-[3H]Phe receptor binding was potentiated by GDP at low concentrations but not by GTP[S]. Furthermore, TD beta gamma GTP[S], but not TD beta gamma GTP or TD beta gamma isolated from TD prepared with Gpp[NH]p (TD beta gamma Gpp[NH]p), prevented fMet-Leu-Phe-stimulated binding of [35S]GTP[S] to G proteins in HL 60 membranes, measured in the presence of GDP. When TD beta gamma GTP was incubated with GTP [S] and TD-depleted illuminated ROS membranes, and subsequently separated from the membranes and free GTP[S], this TD beta gamma GTP, similar to TD beta gamma GTP[S], abolished high-affinity binding of fMet-Leu-[3H]Phe to its receptor, fMet-Leu-Phe-stimulated binding of [35S]GTP[S], and fMet-Leu-Phe-stimulated GTP hydrolysis in HL 60 membranes. Inhibition of [35S]GTP[S] binding by TD beta gamma was not seen in the presence of the metabolically stable GDP analog, guanosine 5'-[beta-thio]diphosphate. In order to obtain an insight into the modification of TD beta gamma apparently caused by GTP[S], and into its mechanism of action in HL 60 membranes, TD, TD alpha and TD beta gamma, all prepared in the presence of GTP, were incubated with [35S]GTP[S] and TD-depleted illuminated ROS membranes. Fluorographic analysis of the supernatant proteins revealed 35S labelling of the beta band of the G protein. When apparently thiophosphorylated TD beta gamma was incubated with [3H]GDP in the presence of HL 60 membranes, [3H]GTP[S] was rapidly formed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solubilization of human placental tumor necrosis factor receptors as a complex with a guanine nucleotide-binding protein.

Human placental membranes exhibited high-affinity receptors for tumor necrosis factor (TNF) (Kd = 5.6 x 10(-10) M) with a density of 1.2-1.7 x 10(10) sites/mg protein. The receptors were solubilized from these membranes with 1% Nonidet P-40, and the solubilized receptor was adsorbed to Con A-Sepharose and wheat germ agglutinin agarose columns, indicating that the TNF receptor derived from human placenta contains carbohydrate chains recognized by these lectins. TNF binding activity was eluted from a column of Sephacryl S-300 as a single peak of Mr 300 kDa. The solubilized receptor was further purified by TNF-Sepharose prepared by coupling of TNF to tresyl-activated Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified sample resolved five major bands of Mr 90, 78, 41, 35, and 11 kDa, suggesting that these polypeptides constitute a multimeric complex with a molecular mass of 300 kDa, as observed in gel filtration study. Furthermore, the TNF-Sepharose-bound fraction demonstrated GTP gamma S binding and GTPase activity. Immunoblot analysis showed that the 41- and 35-kDa polypeptides were recognized by antisera against alpha subunits and beta subunit of GTP-binding proteins, respectively. These results suggest that the native TNF receptor couples to a guanine nucleotide-binding protein to form a large complex structure in human placental membranes.     

An endogenous Ca2+-sensitive proteinase converts the hepatic alpha 1-adrenergic receptor to guanine nucleotide-insensitive forms

An iodoazido[125I]prazosin analogue was employed to photoaffinity label alpha 1-adrenergic receptors in rat liver plasma membranes. Labeled proteins were separated by gradient polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and (-)-epinephrine displacement of [3H]prazosin binding was concurrently measured in the presence or absence of guanosine 5'-O-(gamma-thiotriphosphate) (GTP[gamma S]). Inclusion of EGTA and/or proteinase inhibitors during membrane preparation and incubation increased the effect of GTP[gamma S] on alpha 1-adrenergic agonist binding and this could be correlated with increased concentrations of a 78 kDa photoaffinity labeled protein. In contrast, omission of EGTA or addition of exogenous Ca2+ diminished or abolished the effect of GTP[gamma S] on binding and caused loss of the 78 kDa form and the appearance of lower molecular weight labeled proteins. Age-dependent differences in GTP[gamma S] effects on alpha 1-adrenergic agonist binding were abolished when membranes were prepared and incubated in the presence of EGTA and proteinase inhibitors. However, the 78 kDa photoaffinity labeled protein observed in adult rats (over 225 g body weight) was not apparent in membranes from younger rats (50-75 g), even when the membranes were prepared and incubated in the presence of EGTA and proteinase inhibitors. Instead, a 68 kDa species was the major labeled protein. These data suggest that GTP effects on alpha 1-adrenergic agonist binding in rat liver membranes require the presence of either a 68 or 78 kDa alpha 1-adrenergic binding protein. Failure to inhibit proteolysis in the membranes leads to the generation of lower-molecular-weight binding proteins and the loss of GTP effects on alpha 1-adrenergic agonist binding, although [3H]prazosin binding characteristics are not changed. It is suggested that either the proteolyzed forms of the alpha 1-adrenergic receptor are unable to couple to a putative guanine nucleotide-binding regulatory protein, or that such a protein is concurrently proteolyzed and is thus unable to couple to the receptor.     

Pertussis toxin inhibits cAMP surface receptor-stimulated binding of [35S]GTP gamma S to Dictyostelium discoideum membranes

GTP-binding activity to Dictyostelium discoideum membranes was investigated using various guanine nucleotides. Rank order of binding activities was: GTP gamma S greater than GTP greater than 8-N3-GTP; the binding of GTP gamma S and GTP, but not of 8-N3-GTP, was stimulated by receptor agonists. [3H]GTP binding to D. discoideum membranes has been described previously by a single binding type (Kd = 2.6 microM, Bmax = 85 nM). More detailed studies with [35S]GTP gamma S showed heterogeneous binding composed of two forms of binding sites with respectively high (Kd = 0.2 microM) and low (Kd = 6.3 microM) affinity. cAMP derivatives enhanced GTP gamma S binding by increasing the affinity and the number of the high-affinity sites, while the low-affinity sites were not affected by cAMP. The specificity of cAMP derivatives for stimulation of GTP gamma S binding showed a close correlation with the specificity for binding to the cell surface cAMP receptor. Pretreatment of D. discoideum cells with pertussis toxin did not affect basal GTP and GTP gamma S binding, but eliminated the cAMP stimulation of GTP and GTP gamma S binding. These results indicate that D. discoideum cells have a pertussis toxin-sensitive GTP-binding protein that interacts with the surface cAMP receptor, suggesting the functional interaction of surface receptor with a G-protein in D. discoideum.     

Solubilization of somatostatin receptors in hamster pancreatic beta cells. Characterization as a glycoprotein interacting with a GTP-binding protein

Somatostatin receptors of plasma membranes from beta cells of hamster insulinoma were covalently labelled with 125I-[Leu8,D-Trp22,Tyr25]somatostatin-28 (125I-somatostatin-28) and solubilized with the non-denaturing detergent Triton X-100. Analysis by SDS/PAGE and autoradiography revealed three specific 125I-somatostatin-28 receptor complexes with similar molecular masses (228 kDa, 128 kDa and 45 kDa) to those previously identified [Cotroneo, P., Marie, J.-C. & Rosselin, G. (1988) Eur. J. Biochem. 174, 219-224]. The major labelled complex (128 kDa) was adsorbed to a wheat-germ-agglutinin agarose column and eluted by N-acetylglucosamine. Also, the binding of 125I-somatostatin-28 to plasma membranes was specifically inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate) (GTP[S]) in a dose-dependent manner. Furthermore, when somatostatin-28 receptors were solubilized by Triton X-100 as a reversible complex with 125I-somatostatin-28, GTP[S] specifically dissociated the bound ligand to a larger extent from the soluble receptors than from the plasma-membrane-embedded receptors, the radioactivity remaining bound after 15 min at 37 degrees C being 30% and 83% respectively. After pertussis-toxin-induced [32P]ADP-ribosylation of pancreatic membranes, a 41-kDa [32P]ADP-ribose-labelled inhibitory guanine nucleotide binding protein coeluted with the 128-kDa and 45-kDa receptor complexes. The labelling of both receptor proteins was sensitive to GTP[S]. The labelling of the 228-kDa band was inconsistent. These results support the conclusion that beta cell somatostatin receptors can be solubilized as proteins of 128 kDa and 45 kDa. The major labeled species corresponds to the 128-kDa band and is a glycoprotein. The pancreatic membrane contains a 41-kDa GTP-binding protein that can complex with somatostatin receptors.     

Solubilization of the vasopressin receptor from rat liver plasma membranes. Evidence for a receptor X GTP-binding protein complex

The GTPase activity of plasma membranes isolated from rat livers was stimulated 20% over basal by vasopressin. A concentration dependency curve showed that maximal stimulation was obtained with 10(-8) M vasopressin. The vasopressin-stimulated GTPase activity was not inhibited in plasma membranes that had been ADP-ribosylated with either cholera toxin or pertussis toxin. Identical results were obtained from plasma membranes that had been solubilized with 1% digitonin. When membranes that had been solubilized after preincubation with [3H]vasopressin were subjected to sucrose gradient centrifugation, the majority of protein-bound [3H]vasopressin migrated as a single band with a sedimentation constant of 16.8 S. Moreover, there was a GTPase activity that migrated with the bound [3H]vasopressin. This peak of bound [3H]vasopressin was decreased by 90% when the sucrose gradient centrifugation was run in the presence of 10 microM guanosine 5'-O-(thiotriphosphate). When the 16.8 S peak of bound [3H]vasopressin was further purified over a wheat germ lectin-Sepharose column, a GTPase activity co-eluted from the column with the protein-bound [3H]vasopressin. Direct evidence that a GTP-binding protein was present in the 16.8 S peak was obtained by the immunodetection of a 35-kDa beta subunit of a GTP-binding protein. These results support the conclusion that liver plasma membranes contain a GTP-binding protein that can complex with the vasopressin receptor.     

Reconstitution of the rat liver vasopressin receptor coupled to guanine nucleotide-binding proteins

The V1 vasopressin receptor has been solubilized from rat liver membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammoniol]-1-propanesulfonate (CHAPS) and reconstituted into phospholipid vesicles. There is essentially complete solubilization of the receptor by 3% CHAPS at a protein concentration of 15 mg/ml. Reconstitution into soybean phospholipid vesicles is readily achieved either by gel filtration chromatography or by membrane dialysis. The binding of [3H]vasopressin to proteoliposomes is specific, saturable, reversible, and magnesium-dependent. In contrast, the detergent-soluble vasopressin receptor does not display specific binding. The apparent affinity of the reconstituted receptor for [3H]vasopressin is approximately 4-fold lower than that of the receptor in native membranes. In addition, the binding of [3H]vasopressin to reconstituted vesicles is not sensitive to 100 microM guanosine 5'-O-thiotriphosphate (GTP gamma S) as it is in native membranes. However, the apparent affinity of the reconstituted receptor for ligand approximates that of native membranes when membranes are prebound with vasopressin prior to solubilization and reconstitution into vesicles. Furthermore, vesicles reconstituted from membranes prebound with vasopressin show GTP gamma S sensitivity of [3H] vasopressin binding. This finding strongly suggests that vasopressin stabilizes a receptor-G-protein complex during solubilization. The rat liver vasopressin receptor is a glycoprotein, as shown by its specific binding to the lectin "wheat germ agglutinin." The vasopressin receptor can be reconstituted from the N-acetylglucosamine-eluted peak of a wheat germ agglutinin-Sepharose column, and [3H] vasopressin binding activity is purified 5-6-fold from membranes by this chromatographic procedure. The functionality of the partially purified receptor is indicated by its ability to bind ligand with high affinity and by its ability to functionally interact with a G-protein when vasopressin is bound prior to solubilization.     

Purification of opioid receptor in the presence of sodium ions.

Opioid receptor was solubilized from rat brain membranes with a mixture of the detergents CHAPS and digitonin in the presence of protease inhibitors and 1 M NaCl. The solubilized receptor bound mu-opioid agonists and antagonists with affinities similar to those of native membrane receptor. The affinity of solubilized receptor for the agonist PL017 was greatly reduced by GTP gamma S, suggesting the receptor is still associated with G-protein. The solubilized material was passed through an opioid antagonist (10cd) affinity column and a wheat germ agglutinin column, set up in series, to obtain a partially purified receptor preparation. This partially purified material bound mu-agonist with low affinity and the binding affinity was no longer affected by GTP gamma S. The partially purified receptor was further purified by repeating the affinity and lectin chromatography with smaller size column. Binding of opioid antagonist [3H]diprenorphine to the partially or purified receptors was dependent upon the presence of sodium ions. The purified receptor showed saturable and stereospecific binding for opioid ligands, was predominantly of the mu-type, and exhibited as a diffuse band with a medium molecular mass of 62 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The average specific binding activity of the purified receptor was 18.8 +/- 2.3 pmol/micrograms protein, a value close to the theoretical estimation.     

A prostaglandin E receptor coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein in rabbit cortical collecting tubule cells

At different concentrations, prostaglandin E2 (PGE2) can either stimulate or inhibit cAMP formation in freshly isolated rabbit cortical collecting tubule (RCCT) cells, but in cultured RCCT cells PGE2 can only stimulate cAMP synthesis (Sonnenburg, W. K., and Smith W. L. (1989) J. Biol. Chem. 263, 6155-6160). Here, we report characteristics of [3H]PGE2 binding to membrane receptor preparations from both freshly isolated and cultured RCCT cells. [3H]PGE2 binding to membranes from freshly isolated RCCT cells was saturable and partially reversible. Equilibrium binding analyses indicated that in the absence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) there is a single class of PGE2 binding sites (KD = 4.2 +/- 0.4 nM; Bmax = 583 +/- 28 fmol/mg); in the presence of 100 microM GTP gamma S, there is also only one class of binding sites but with a somewhat lower KD = 1.2 +/- 0.5 nM (Bmax = 370 +/- 40 fmol/mg). This stimulatory effect of GTP gamma S was blocked by pretreatment of the freshly isolated RCCT cells with pertussis toxin. The relative affinities of prostanoids for the [3H]PGE2-binding site were determined to be 17,18,19,20-tetranor-16-phenoxy-PGE2-methylsulfonylamide (sulprostone) approximately PGE2 approximately PGE1 approximately 16,16-dimethyl-PGE2 greater than carbacyclin approximately PGF2 alpha greater than PGD2. This is the order of potency with which prostaglandins inhibit arginine vasopressin-induced cAMP formation in fresh RCCT cells. Interestingly, [3H]PGE2 binding to membranes from cultured cells, which, unlike fresh cells, fail to show an inhibitory response to PGE2, was only 10-20% of that observed with membranes from fresh cells; moreover, binding of [3H]PGE2 to membranes from cultured cells was neither stimulated by GTP gamma S nor inhibited by sulprostone. The prostanoid binding specificities and the unusual pertussis toxin-sensitive, stimulatory effect of GTP gamma S on binding of [3H]PGE2 to membranes from freshly isolated RCCT cells are characteristics shared by a Gi-linked PGE receptor from renal medulla (Watanabe, T., Umegaki, K., and Smith, W. L. (1986) J. Biol. Chem. 261, 14340-14349). Our results suggest that the [3H]PGE2 binding site of freshly isolated RCCT cells is the PGE receptor which is coupled to a Gi to attenuate arginine vasopressin-induced cAMP synthesis in the renal collecting tubule.     

The formylpeptide chemoattractant receptor copurifies with a GTP-binding protein containing a distinct 40-kDa pertussis toxin substrate

Detergent extraction of plasma membranes from differentiated HL60 cells, specifically labeled with the chemoattractant, formyl-Nle-Leu-Phe-Nle-[125I-Tyr] Lys, resulted in the solubilization of a receptor-radioligand complex. GTP-binding activity coeluted with the radioligand when the sodium cholate extract was purified by chromatography on wheat germ agglutinin-Sepharose 6MB. A molecular size of approximately 59 A was estimated for the lectin-Sepharose-purified receptor complex by gel filtration chromatography on Ultrogel AcA 34. The isolated complex eluted from the gel filtration column exhibited an enhanced rate of ligand dissociation in response to GTP gamma S. Approximately 0.65 mol of pertussis toxin substrate/mol of receptor was estimated following partial purification of the receptor-ligand complex by sequential chromatography on wheat germ agglutinin-Sepharose, DEAE-Fractogel, and Ultrogel AcA 34. The pertussis toxin substrate which copurified with the receptor was compared with two distinct G proteins, containing alpha-subunits of 40 and 41 kDa, previously purified from HL60 cell plasma membranes. Approximately 86% of the pertussis toxin substrate identified in the receptor preparation consisted of the 40-kDa polypeptide. Differences in the peptide maps indicate that the predominant G protein which coelutes with the receptor is distinct from the purified G protein with an alpha-subunit of 41 kDa but homologous to the purified G protein with an alpha-subunit of 40 kDa.     

Solubilization and purification of the prostaglandin E2 receptor from cardiac sarcolemma.

A prostaglandin E2 (PGE2) receptor was solubilized and isolated from cardiac sarcolemma membranes. Its binding characteristics are almost identical to those of the membrane bound receptor. [3H]PGE2 binding to solubilized and membrane bound receptor was sensitive to elevated temperature and no binding was observed in the absence of NaCl. No significant effects of DTT, ATP, Mg2+, Ca2+ or of changes in buffer pH were observed on [3H]PGE2 binding to either solubilized or membrane-bound receptor. Unlabelled PGE1 displaced over 90% of [3H]PGE2 from the CHAPS-solubilized receptor. PGD2, PGI2, PGF2 alpha and 6-keto-PGF1 alpha were not effective in displacing [3H]PGE2 from the receptor. Scatchard analysis of [3H]PGE2 binding to CHAPS-solubilized receptor revealed the presence of two types of PGE2 binding sites with Kd of 0.33 +/- 0.05 nM and 3.00 +/- 0.27 nM and Bmax of 0.5 +/- 0.04 and 2.0 +/- 0.1 pmol/mg of protein. The functional PGE2 receptor was isolated from CHAPS-solubilized SL membrane using two independent methods: first by a WGA-Sepharose chromatography and second by sucrose gradient density centrifugation. Receptor isolated by these two methods bound [3H]PGE2. Unlabelled PGE1 and PGE2 displaced [3H]PGE2 from the purified receptor. Scatchard analysis of [3H]PGE2 binding to purified receptor revealed the presence of the two binding sites as observed for the membrane bound and CHAPS-solubilized receptor. SDS-polyacrylamide gel electrophoresis of the purified receptor fractions revealed the presence of a protein band of M(r) of approx. 100,000. This 100-kDa was photolabelled with [3H]azido-PGE2, a photoactive derivative of PGE2. We propose that this 100-kDa protein is a cardiac PGE2 receptor.     

Characterization and site-directed mutagenesis of a low M(r) GTP-binding protein, ram p25, expressed in Escherichia coli.

The ram gene encodes a GTP-binding protein with a M(r) of 25,068 (Nagata, K., Satoh, T., Itoh, H., Kozasa, T., Okano, Y., Doi, T., Kaziro, Y., and Nozawa, Y. (1990) FEBS Lett. 275, 29-32). It has a putative effector domain very similar to that of yeast SEC4 protein, and shares 40% identity and 60% homology with it, respectively. In order to analyze the biochemical properties, ram cDNA was engineered and inserted into a bacterial expression vector; this allowed the production at a high level of soluble recombinant ram p25 in Escherichia coli. The purified ram p25 contained an equimolar amount of GDP. The purified protein bound approximately 1 mol of [35S]guanosine 5'-O-(thiotriphosphate) GTP gamma S)/mol of protein, with a Kd value of 120 nM. [35S]GTP gamma S binding to this protein was inhibited by GTP and GDP, but not by ATP and ADP. In the presence of 10 mM Mg2+, the dissociation of [8,5'-3H]GDP and [35S]GTP gamma S from ram p25 occurred with rates of 0.015 min-1 and 0.004 min-1, respectively, showing that the ram p25 has a higher affinity for GTP than GDP. The rate of release of Pi from [gamma-32P]GTP-bound ram p25 was calculated to be 0.011 min-1. The contribution of guanine nucleotide-binding and GTP-hydrolysis domains of the protein to its biochemical activities was investigated by site-directed mutagenesis. Substitution of Val for Gly at position 19 resulted in disappearance of [35S]GTP gamma S- and [3H]GDP-binding activity in spite of good expression of the protein. Mutations of Thr41 to Ser, Ala76 to Thr, and Asn133 to His slightly increased the rates of [35S] GTP gamma S binding and [3H]GDP dissociation, but had almost no effects on the manner of [gamma-32P]GTP hydrolysis. Replacement of Gln78 with Leu significantly increased the [3H]GDP dissociation rate (7-fold) and decreased GTP hydrolytic activity considerably.     

Phosphorylation by cyclic AMP-dependent protein kinase of a human platelet Mr 22,000 GTP-binding protein (smg p21) having the same putative effector domain as the ras gene products

We have purified to near homogeneity a Mr 22,000 GTP-binding protein from human platelet membranes and identified it as the smg-21 gene product (smg p21), having the same putative effector domain as the ras gene products, which we have purified to near homogeneity from bovine brain membranes and characterized. This purified human platelet smg p21 was phosphorylated by cyclic AMP-dependent protein kinase. About one mol of phosphate was maximally incorporated into one mol of the protein. Only serine residue was phosphorylated. Both the guanosine 5'-(3-O-thio)-triphosphate (GTP gamma S)-bound and GDP-bound forms were phosphorylated with the same reaction velocity. The phosphorylation of smg p21 affected neither its GTP gamma S-binding nor GTPase activity. Human platelet smg p21 was not phosphorylated by protein kinase C. A Mr 24,000 GTP-binding protein partially purified from human platelet membranes was not phosphorylated by cyclic AMP-dependent protein kinase or protein kinase C.     

GTP-binding proteins in luminal and basolateral membranes from pars convoluta and pars recta of rabbit kidney proximal tubule.

The GTP-binding proteins on luminal and basolateral membrane vesicles from outer cortex (pars convoluta) and outer medulla (pars recta) of rabbit proximal tubule have been examined. The membrane vesicles were highly purified, as ascertained by electron microscopy, by measurements of marker enzymes, and by investigating segmental-specific transport systems. The [35S]GTP gamma S binding to vesicles, and to sodium cholate-extracted proteins from vesicles, indicated that the total content of GTP-binding proteins were equally distributed on pars convoluta, pars recta luminal and basolateral membranes. The membranes were ADP-ribosylated with [32P]NAD+ in the presence of pertussis toxin and cholera toxin. Gel electrophoresis revealed, for all preparations, the presence of cholera toxin [32P]ADP-ribosylated 42 and 45 kDa G alpha s proteins, and pertussis toxin [32P]ADP-ribosylated 41 kDa G alpha i1, 40 kDa G alpha i2 and 41 kDa G alpha i3 proteins. The 2D electrophoresis indicated that Go's were not present in luminal nor in basolateral membranes of pars convoluta or pars recta of rabbit proximal tubule.