Multiple affinity states of opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells. Opiate agonist association rate is a function of receptor occupancy

The existence of multiple affinity states for the opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells has been demonstrated by competition binding studies with tritiated diprenorphine and [D-Ala2, D-Leu5]enkephalin (DADLE). In the presence of 10 mM Mg2+, all receptors exist in a high affinity state with Kd = 1.88 +/- 0.16 nM. Addition of 10 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) decreased the affinity of DADLE to Kd = 8.08 +/- 0.93 nM. However, in the presence of 100 mM Na+, which is required for opiate inhibition of adenylate cyclase activity, analysis of competition binding data revealed three sites: the first, consisting of 17.5% of total receptor population has a Kd = 0.38 +/- 0.18 nM; the second, 50.6% of the population, has a Kd = 6.8 +/- 2.2 nM; and the third, 31.9% of the population, has a Kd of 410 +/- 110 nM. Thus, in the presence of sodium, a high affinity complex between receptor (R), GTP binding component (Ni), and ligand (L) was formed which was different from that formed in the absence of sodium. These multiple affinity states of receptor in the hybrid cells are agonist-specific, and the percentage of total opiate receptor in high affinity state is relatively constant in various concentrations of Na+. Multiple affinity states of opiate receptor can be demonstrated further by Scatchard analysis of saturation binding studies with [3H]DADLE. In the presence of Mg2+, or Gpp(NH)p, analysis of [3H]DADLE binding demonstrates that opiate receptor can exist in a single affinity state, with apparent Kd values of [3H]DADLE in 10 mM Mg2+ = 1.75 +/- 0.28 nM and in 10 microM Gpp(NH)p = 0.85 +/- 0.12 nM. There is a reduction of Bmax value from 0.19 +/- 0.02 nM in the presence of Mg2+ to 0.14 +/- 0.03 nM in the presence of Gpp(NH)p. In the presence of 100 mM Na+, Scatchard analysis of saturation binding of [3H]DADLE reveals nonlinear plots; two-site analysis of the curves yields Kd = 0.43 +/- 0.09 and 7.9 +/- 3.2 nM. These Kd values are analogous to that obtained with competition binding studies. Again, this conversion of single site binding Scatchard plots to multiple sites binding plots in the presence of Na+ is restricted to 3H-agonist binding only.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of γ-Aminobutyric Acid Binding Sites on Crude Synaptic Membranes

[3H]GABA binding to crude synaptic membranes of rat brain was studied in an attempt to identify GABA binding to its synaptic receptor in the presence of Na+. Membrane vesicles prepared from crude synaptic membrane fractions were useful as a tool to differentiate synaptic GABA receptors from GABA uptake sites. The crude synaptic membranes treated with Triton X-100 [membranes (TX)] involved two classes of GABA binding sites (KD = 38.7 and 78.0 nM) in the absence of Na+, but the high-affinity sites disappeared in the presence of Na+ and a single class of GABA binding sites (KD = 75.0 nM) was detected. The failure to detect an active uptake of [3H]GABA into the vesicles prepared from membranes (TX) suggests that the [3H]GABA binding in the presence of Na+ was related to synaptic GABA receptors. It is probable that Na+ could mask the presence of the high-affinity class of GABA receptor.     

Regulation of thyrotropin-releasing hormone binding by monovalent cations and guanyl nucleotides

Binding of thyrotropin-releasing hormone (TRH) to specific receptors on membranes isolated from GH4C1 pituitary cells was inhibited by monovalent cations and guanyl nucleotides. NaCl and LiCl inhibited TRH binding by 70%, with half-maximal inhibition at 30 mM; RbCl and KCl inhibited only 10% at concentrations up to 150 mM. NaCl decreased both the apparent number and the affinity of TRH receptors and increased the rate of dissociation of TRH from both membrane and Triton X-100-solubilized receptors. Guanyl nucleotides inhibited TRH binding up to 80%, with guanyl-5'-yl imidodiphosphate (Gpp(NH)p) approximately GTP much greater than GDP approximately ATP greater than GMP. GTP and Gpp(NH)p exerted half-maximal effects at 0.3 microM and decreased receptor affinity to one-third of control but did not change receptor number. Gpp(NH)p accelerated the dissociation of TRH from membranes but not from solubilized receptors. The effects of NaCl were independent of temperature, while GTP and Gpp(NH)p were much more inhibitory at 22 degrees C (70%) than at 0 degrees C (10%). Inhibition by NaCl could be reversed by washing the membranes, and inhibition by GTP was reversed if membranes were chilled to 0 degrees C. The inhibitory effects of low concentrations of NaCl and Gpp(NH)p were additive. Neither monovalent cations nor GTP prevented the TRH-receptor complex from undergoing transformation from a state with rapid dissociation kinetics to a slower dissociating form. The results suggest that sodium ion regulates TRH binding by interacting with a site on the receptor, while guanyl nucleotides regulate TRH binding indirectly.     

Opiate Binding Sites in Bovine Adrenal Medulla

Abstract

Previous studies using a variety of opiate ligands have suggested the existence of several subclasses of opiate receptors in crude membrane fractions of rat brain, and a similar diversity in bovine adrenal medulla. To examine the receptor profile of bovine adrenal medulla in detail we have studied the binding of classical ligands for mu (μ), delta (δ) and kappa (k) opiate receptors. [³H]naloxone ([³H]NAL), [³H] morphine ([³H]MOR), [³H]D-Ala²-D-Leu⁵-enkephalin ([³H]DAL) and [³H]ethyl-ketocyclazocine ([³H]EKCZ) were used as tracers; unlabeled competitors were NAL, MOR, DAL and ketocyclazocine (KCZ). In adrenal medulla [³H]NAL was specifically bound with a hierarchy of displacement NAL > MOR > KCZ ? DAL. No specific binding of [³H]DAL or [³H]EKCZ was found; for [³H]MOR very low levels of binding were seen, with no displacement by NAL or DAL, inconsistent displacement by KCZ and substantial displacement by MOR with an ED₅₀ of 1.5 nM. In parallel studies rat brain membranes bound each labeled ligand with affinity and specificity consistent with previously published reports. Identical results were obtained in membranes from both tissues prepared with a preincubation step including 100 mM Na⁺, suggesting that the results were not influenced by occupation of binding sites by endogenous ligands. We interpret these data as supporting the existence of opiate receptors of the μ subtype in bovine adrenal medulla. We find, however, no evidence of δ or k sites in this tissue.     

Enhancement of [3H]DAGO1 binding to rat brain by low concentrations of monovalent cations

The effects of mono- and di-valent cations and the nonhydrolyzable guanyl nucleotide derivative 5'-guanylimidodiphosphate (Gpp(NH)p) on the binding of the selective, high affinity mu-opiate receptor agonist, [3H]DAGO ([3H]Tyr-D-Ala-Gly-Mephe-Gly-ol), to rat brain membranes were studied in a low ionic strength 5 mM Tris-HCl buffer. Na+ and Li+ (50 mM) maximally increased [3H]DAGO binding (EC50 values for Na+, 2.9 mM and Li+, 6.2 mM) by revealing a population of low affinity binding sites. The density of high affinity [3H]DAGO binding sites was unaffected by Na+ and Li+, but was maximally increased by 50 mM K+ and Rb+ (EC50 values for K+, 8.5 mM and Rb+, 12.9 mM). Divalent cations (Ca2+, Mg2+; 50 mM) inhibited [3H]DAGO binding. Gpp(NH)p decreased the affinity of [3H]DAGO binding, an effect that was enhanced by Na+ but not by K+. The binding of the mu-agonist [3H]dihydromorphine was unaffected by 50 mM Na+ in 5 mM Tris-HCl. In 50 mM Tris-HCl, Na+ (50 mM) inhibited [3H]DAGO binding by decreasing the density of high affinity binding sites and promoting low affinity binding. The effects of Na+ in 5 mM and 50 mM Tris-HCl were also investigated on the binding of other opiate receptor agonists and antagonists. [3H]D-Ala-D-Leu-enkephalin binding was increased and inhibited. [3H]etorphine binding increased and was unchanged, and both [3H]bremazocine and [3H]naloxone binding increased by 50 mM Na+ in 5 mM and 50 mM Tris-HCl, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity labeling of delta-opiate receptors using [D-Ala2,Leu5,Cys6]enkephalin. Covalent attachment via thiol-disulfide exchange

[D-Ala2,Leu5,Cys6]Enkephalin (DALCE) is a synthetic enkephalin analog which contains a sulfhydryl group. DALCE binds with high affinity to delta-receptors, with moderate affinity to mu-receptors, and with negligible affinity to kappa-receptors. Pretreatment of rat brain membranes with DALCE resulted in concentration-dependent loss of delta-binding sites. Using 2 nM [3H][D-Pen2,D-Pen5]enkephalin (where Pen represents penicillamine) to label delta-sites, 50% loss of sites occurred at about 3 microM DALCE. Loss of sites was not reversed by subsequent incubation in buffer containing 250 mM NaCl and 100 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p), conditions which cause dissociation of opiate agonists. By contrast, the enkephalin analogs [D-Ala2,D-Leu5]enkephalin, [D-Ser2,Leu5,Thr6]enkephalin, [D-Pen2,D-Pen5]enkephalin, and [D-Ala2,D-Leu5,Lys6]enkephalin were readily dissociated by NaCl and Gpp(NH)p, producing negligible loss at 3 microM. This suggests that DALCE binds covalently to the receptors. Pretreatment of membranes with the reducing agents dithiothreitol and beta-mercaptoethanol had no effect on opiate binding. Thus, loss of sites required both specific recognition by opiate receptors and a thiol group. The irreversible effect of DALCE was completely selective for delta-receptors. Pretreatment with DALCE had no effect on binding of ligands to mu- or kappa-receptors. The effect of DALCE on delta-binding was: 1) markedly attenuated by inclusion of dithiothreitol in the preincubation buffer, 2) partially reversed by subsequent incubation with dithiothreitol, 3) slightly enhanced when converted to the disulfide-linked dimer, and 4) prevented by blocking the DALCE sulfhydryl group with N-ethylmaleimide or iodoacetamide. These results indicate that DALCE binds covalently to delta-receptors by forming a disulfide bond with a sulfhydryl group in the binding site. The mechanism may involve a thiol-disulfide exchange reaction.     

Binding of [3H]forskolin to human platelet membranes. Regulation by guanyl-5'-yl imidodiphosphate, NaF, and prostaglandins E1 and D2

[3H]Forskolin binds to human platelet membranes in the presence of 5 mM MgCl2 with a Bmax of 125 fmol/mg of protein and a Kd of 20 nM. The Bmax for [3H]forskolin binding is increased to 455 and 425 fmol/mg of protein in the presence of 100 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and 10 mM NaF, respectively. The increase in the Bmax for [3H]forskolin in the presence of Gpp(NH)p or NaF is not observed in the absence of MgCl2. The EC50 values for the increase in the number of binding sites for [3H]forskolin by Gpp(NH)p and NaF are 600 nM and 4 mM, respectively. The EC50 value for Gpp(NH)p to increase the number of [3H]forskolin binding sites is reduced to 35 mM and 150 nM in the presence of 50 microM PGE1 or PGD2, respectively. The increase in the number of [3H]forskolin binding sites observed in the presence of NaF is unaffected by prostaglandins. The binding of [3H]forskolin to membranes that are preincubated with Gpp(NH)p for 120 min or assayed in the presence of PGE1 reaches equilibrium within 15 min. In contrast, a slow linear increase in [3H]forskolin binding is observed over a period of 60 min when Gpp(NH)p and [3H]forskolin are added simultaneously to membranes. A slow linear increase in adenylate cyclase activity is also observed as a result of preincubating membranes with Gpp(NH)p. In human platelet membranes, agents that activate adenylate cyclase via the guanine nucleotide stimulatory protein (Ns) increase the number of binding sites for [3H]forskolin in a magnesium-dependent manner. This is consistent with the high affinity binding sites for [3H]forskolin being associated with the formation of an activated complex of the Ns protein and adenylate cyclase. This state of the adenylate cyclase may be representative of that formed by a synergistic combination of hormones and forskolin.     

Differential Regulation by Guanine Nucleotides of Opiate Agonist and Antagonist Receptor Interactions

Abstract: Guanine nucleotides differentiate binding of tritium-labeled agonists and antagonists to rat brain membranes. In the absence of sodium, GTP (50 μM) decreased binding of [³H]-labeled agonists by 20–60% and [³H]-labeled antagonists by 0–20%. In the presence of 100 mM-NaCl, GTP had no effect on antagonist binding, but decreased agonist binding by 60–95%. GMP was less potent than either GTP or GDP in decreasing agonist binding. GTP (50 μM) reduced high-affinity [³H]dihydromorphine sites by 52% and low-affinity sites by 55%. Without sodium, GTP reduced high-affinity [³H]-naloxone sites by 36%; in the presence of 100 mM-NaCl, GTP had no effect on either high- or low-affinity [³H]naloxone sites. GTP increased the association rate of [³H]dihydromorphine twofold and the dissociation rate by fourfold, while having no effect on association or dissociation rates of the antagonist [³H]diprenorphine. The affinities of uniabeled antagonists in inhibiting [³H]-diprenorphine binding were not affected by GTP or sodium, but the affinities of agonists were reduced 40- 120-fold, with met- and leu-enkephalin affinities reduced by the greatest degree. GTP and sodium lowered [³H]dihydromorphine binding in an additive fashion, while divalent cations, especially manganese, reversed the effects of GTP on [³H]-labeled agonist binding by stimulating membrane-bound phosphatases that hydrolyze GTP to GMP and guanosine. These results suggest that by affecting binding of agonists, but not antagonists, GTP may regulate opiate receptor interactions with their physiological effectors.     

Receptor binding and G-protein activation by new Met5-enkephalin-Arg6-Phe7 derived peptides

Met5-enkephalin-Arg6-Phe7 (Tyr-Gly-Gly-Phe-Met-Arg-Phe, MERF) is a naturally occurring heptapeptide that binds to opioid and non-opioid recognition sites in the central nervous system. Four synthetic analogs with single or double amino acid substitutions were prepared by solid phase peptide synthesis to achieve proteolytically more stable structures: Tyr-D-Ala-Gly-Phe-Met-Arg-Phe (I), Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (II), Tyr-D-Ala-Gly-Phe-L-Nle-Arg-Phe (III) and Tyr-Gly-Gly-Phe-L-Nle-Arg-Phe (IV). In this study receptor binding characteristics and G-protein activation of MERF and its derivatives were compared in crude membrane fractions of frog and rat brain. Synthetic MERF-derived peptides were potent competitors for [3H]MERF and [3H]naloxone binding sites with the exception of analog (II) which turned to be substantially less active. The presence of 100 mM NaCl or 100 microM 5'-guanylylimidodiphosphate, Gpp(NH)p, decreased the affinity of the peptides in [3H]naloxone binding assays, suggesting that these ligands might act as agonists at the opioid receptors. Some of the compounds were also used to stimulate guanosine-5'-O-(3-[gamma-[35S]thio)triphosphate ([35S]GTPgammaS) binding in rat and frog brain membranes at concentrations of 10(-9)-10(-5) M. The EC50 values of analog (II) were the highest in both tissues. Analog (I) was as effective as MERF in rat brain membranes, but showed lower maximal stimulation in frog brain preparation. Again, analog (II) seemed to be the least efficacious peptide that stimulated [35S]GTPgammaS binding only by 59%. Specificity of the peptides was further investigated by the inhibition of agonist-stimulated [35S]GTPgammaS binding in the presence of selective antagonists for the opioid receptor types. The mu-selective antagonist cyprodime displayed the lowest potency in inhibiting the effects of the peptides, whereas norbinaltorphimine (kappa-selective antagonist) and naltrindole (delta-selective antagonist) were quite potent in both tissues. We concluded that MERF and its derivatives are able to activate G-proteins mainly via kappa- and delta-opioid receptors.     

CHAPS solubilization of a G-protein sensitive 5-HT1A receptor from bovine hippocampus

The binding of [3H] 8-OH-DPAT to membrane-bound 5-HT1A receptors from bovine hippocampus was saturable and corresponded to a single high-affinity state. Solubilization of the bovine hippocampal membranes with 10 mM CHAPS containing 200 mM NaCl, renders a preparation which binds [3H] 8-OH-DPAT with high-affinity (Kd = 1.9 nM) and is guanine nucleotide sensitive and ketanserin insensitive. 50% of [3H] 8-OH-DPAT binding activity is solubilized. The presence of GMP-P(NH)P promotes a low-affinity (Kd = 9.6 nM) state which is characteristic of receptors coupled to G-proteins. GMP-P(NH)P markedly accelerates the dissociation [3H] 8-OH-DPAT from solubilized membranes while having negligible effects on association. Thus, the agonist can activate the terniary complex rather than to promote its formation. 8-OH DPAT, WB 4101 and 5-carboxamidotryptamine dose responsively inhibit soluble [3H] 8-OH-DPAT binding with IC(50) values of 16.1, 15.6 and 1.3 nM, respectively. The CHAPS solubilized membrane preparation retains many of the [3H] 8-OH-DPAT binding characteristics of the membrane bound form.     

Opiate binding sites and endogenous opioids in Bufo viridis oocytes

Binding sites with high affinity for [3H]naloxone, but not for [3H]morphine and [3H] (D-Ala2, D-Leu5) enkephalin, have been found in membranes of Bufo viridis oocytes. The binding is reversible and saturable. Bound [3H]naloxone is easily displaced both by unlabeled naloxone and bremazocine, much worse by morphine and SKF 10,047; (D-Ala2, D-Leu5) enkephalin and beta-endorphin practically fail to displace [3H]naloxone. Scatchard analysis is consistent with the existence of two classes of binding sites with Kd 15 nM and 10(3) nM. The number of binding sites with high affinity for naloxone is 16 pmol/mg protein of homogenized oocytes which is 20-50-fold higher than in, toad or rat brain. Oocyte extract displaces [3H]naloxone bound with oocytes' membranes and inhibits electrically evoked contractions of the rabbit vas deferens. This inhibition is reversed by naloxone. It is suggested that compounds similar to opiate kappa-agonists exist in oocytes. It cannot be ruled out that they participate via specific receptors in the regulation of oocyte maturation and egg development.     

pH Selectivity of N-Ethylmaleimide Reactions with Opiate Receptor Complexes in Rat Brain Membranes

N-Ethylmaleimide (NEM) decreases opiate agonist binding presumably by blocking crucial sulfhydryl (SH) groups at receptor binding sites. At physiological pH, NEM decreased GTP and manganese regulation but increased sodium effects on [3H]D-Ala2-Met5-enkephalinamide (D-Ala enk) binding to rat brain membranes. To determine the apparent pK values of putative SH groups in opiate receptors that react with NEM, rat brain membranes were incubated with 100-250 microM NEM in buffers ranging from pH 4.5 to 8.0. Results showed that lowering pH below 6.5 reduced the NEM effect on opiate receptor functions and that the apparent pK values of NEM-reacting SH groups in binding and regulatory sites ranged between 5.4 to 6.0. Most of the total SH groups in brain membranes continued to react with NEM at low pH, so that when nonspecific SH groups were blocked by incubating membranes at pH 4.5 with NEM, opiate receptors became sensitive to very low concentrations (1 microM) of NEM.     

Opiate binding to membrane preparations of neuroblastoma x glioma hybrid cells NG108-15: effects of ions and nucleotides.

Interaction of a number of opiate agonists with the opiate receptors in NG108-15 cell membranes is influenced by ions, as well as certain nucleotides. Steady state binding of [³H]leu-enkephalin is increased by Mg⁺⁺ and decreased by Na⁺, GMP-P(NH)P, GTP, GDP, ITP and IMP-P(NH)P. Half-maximal inhibition produced by GMP-P(NH)P occurred at 4.6 μM. The dissociation of [³H]leu- and [³H]met-enkephalin, as well as [³H]etorphine, from these opiate receptors was also shown to be altered by both ions and nucleotides.     

Exchange of Guanine Nucleotides Between Tubulin and GTP-Binding Proteins That Regulate Adenylate Cyclase: Cytoskeletal Modification of Neuronal Signal Transduction

Tubulin, the primary constituent of microtubules, is a GTP-binding proteins with structural similarities to other GTP-binding proteins. Whereas microtubules have been implicated as modulators of the adenylate cyclase system, the mechanism of this regulation has been elusive. Tubulin, polymerized with the hydrolysis-resistant GTP analog, 5'-guanylylimidodiphosphate [Gpp(NH)p], can promote inhibition of synaptic membrane adenylate cyclase which persists subsequent to washing. Tubulin with Gpp(NH)p bound was slightly less potent than free Gpp(NH)p in the inhibition of adenylate cyclase, but tubulin without nucleotide bound had no effect on the enzyme. A GTP-binding protein from the rod outer segment (transducin), with Gpp(NH)p bound, was also without effect on adenylate cyclase. Tubulin (regardless of the nucleotide bound to it) did not alter the activity of the adenylate cyclase catalytic unit directly. When tubulin was polymerized with the hydrolysis-resistant photoaffinity GTP analog, [32P]P3(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP), and this protein was added to synaptic membranes, AAGTP was transferred from tubulin to the inhibitory GTP-binding protein, Gi. This transfer was blocked by prior incubation of the membranes with Gpp(NH)p or covalent binding of AAGTP to tubulin prior to exposure of that tubulin to membranes. Incubation of membranes with Gpp(NH)p subsequent to incubation with tubulin-AAGTP results in a decrease in AAGTP bound to Gi and a compensatory increase in AAGTP bound to the stimulatory GTP-binding protein, Gs. Likewise, persistent inhibition of adenylate cyclase by tubulin-Gpp(NH)p could be overridden by the inclusion of 100 microM Gpp(NH)p in the assay inhibition. Whereas Gpp(NH)p promotes persistent inhibition of synaptic membrane adenylate cyclase without incubation at elevated temperatures, tubulin [with AAGTP or Gpp(NH)p bound] requires 30 s incubation at 23 degrees C to effect adenylate cyclase inhibition. Photoaffinity experiments yield parallel results. These data are consistent with synaptic membrane tubulin regulating neuronal adenylate cyclase by transferring GTP to Gi and, subsequently, to Gs.     

Complete Conversion of Brain D2 Dopamine Receptors from the High- to the Low-Affinity State for Dopamine Agonists, Using Sodium Ions and Guanine Nucleotide

Since previous work had shown that brain D2 3,4-dihydroxyphenylethylamine (dopamine) receptors were only partly converted from their high-affinity state to their low-affinity state, we here tested whether it was possible to obtain a complete 100% conversion of these receptors into their low-affinity state. It was first essential to resolve the components of [3H]spiperone binding to dopaminergic sites and nondopaminergic sites in rat striatal homogenates. In the presence of 50 microM S-sulpiride (to occlude the dopaminergic sites), therefore, we first determined that the residual binding of [3H]spiperone (approximately 20%) was inhibited by serotonergic agonists much more effectively than dopamine or noradrenaline, thus identifying the serotonergic component of [3H]spiperone binding. Thus, dopamine (or ADTN) inhibited the binding of [3H]spiperone at a high-affinity site (with dissociation constant of 10 nM dopamine), at a low-affinity site (with dissociation constant of 2,000 nM dopamine), and at the serotonergic site (with dissociation constant of 50,000 nM dopamine). In the absence of sodium ions, the high-affinity site was about 50% occupied by [3H]spiperone, and guanine nucleotide had no effect on this proportion. In the presence of 120 mM NaCl, however, the high-affinity site was reduced to 15% and guanine nucleotide completely eliminated this high-affinity site, 100% of the sites having been completely converted to their low-affinity state. Using [3H]N-propyl-norapomorphine to label the high-affinity state of the dopamine receptor, 50% conversion into the low-affinity state occurred at 45 mM LiCl, 69 mM NaCl, and 202 mM KCl. We conclude that it is possible to convert brain D2 dopamine receptors completely into their low-affinity state, in the presence of NaCl and a guanine nucleotide, providing that appropriate allowance is made for the serotonergic component of [3H]spiperone binding.     

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)     

Comparison of the Effects of Ions and GTP on Substance P Binding to Membrane-Bound and Solubilized Specific Sites

Mg2+ increased but Na+ and GTP decrease [3H]substance P (SP) binding to rat cerebral cortical membranes and to 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS)-solubilized membrane fraction. To determine the binding parameters that are modified by the cations and GTP, inhibition experiments of [3H]SP binding by unlabeled SP were performed in both of the preparations. Nonlinear least-squares regression analysis of data in the membrane fraction indicated that optimal fitting of the inhibition curves in the presence of 10 mM MgCl2 was attained with a two-site model, corresponding to a "high-affinity (H)" and a "low-affinity (L)" state. By omitting MgCl2, or by addition of NaCl and GTP, the [3H]SP specific binding was decreased, the H state disappeared, and the L state and a new "super-low affinity (SL)" state observed. The SP/[3H]SP inhibition curves in the cerebral cortical membranes by in vivo treatment with pertussis toxin (islet-activating protein) were similar to that in the presence of GTP in control membranes. The effects of MgCl2, NaCl, and GTP were greater in the CHAPS-solubilized fraction than in the membrane fraction. In contrast to the membrane fraction, the inhibition curves of [3H]SP binding by unlabeled SP in the presence of MgCl2 in the CHAPS-solubilized fraction were best fitted to a one-site model. The KD value was relatively close to that of the low-affinity state in the membrane fraction. Even with the addition of NaCl or GTP, or by reducing MgCl2 concentration to 1 mM, although the inhibition curves consistently fit the one-site model, the KD values changed only slightly.     

Interaction of l-Prolyl-l-Leucyl Glycinamide with Dopamine D2 Receptor: Evidence for Modulation of Agonist Affinity States in Bovine Striatal Membranes

The role of the hypothalamic tripeptide L-prolyl-L-leucyl-glycinamide (PLG) in modulating the agonist binding to bovine striatal dopamine D2 receptor was investigated using a selective high-affinity agonist, n-propylnorapomorphine (NPA). PLG caused an enhancement in [3H]NPA binding in striatal membranes in a dose-dependent manner, the maximum effect being observed at 10(-7)-10(-6) M concentration of the tripeptide. The Scatchard analysis of [3H]NPA binding to membranes preincubated with 10(-6) M PLG revealed a significant increase in the affinity of the agonist binding sites. In contrast, there was no effect of PLG on the binding pattern of the antagonist [3H]spiroperidol. The antagonist versus agonist competition curves analyzed for agonist high- and low-affinity states of the receptor displayed an increase in the population and affinity of the high-affinity form of the receptor with PLG treatment. The low-affinity sites concomitantly decreased with relatively small change in the affinity for the agonists. Almost similar results were obtained when either NPA or apomorphine was used in the competition experiments. A partial antagonistic effect of PLG on 5'-guanylylimidodiphosphate [Gpp(NH)p]-induced inhibition of high-affinity agonist binding was also observed, as the ratio of high- to low-affinity forms of the receptor was significantly higher in the PLG-treated membranes compared to the controls. Direct [3H]NPA binding experiments demonstrated that PLG attenuated the Gpp(NH)p-induced inhibition of agonist binding by increasing the EC50 of the nucleotide (concentration that inhibits 50% of the specific binding). No effect of PLG on high-affinity [3H]NPA binding, however, could be observed when the striatal membranes were preincubated with Gpp(NH)p.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization and reconstitution of dopamine D1 receptor from bovine striatal membranes: Effects of agonist and antagonist pretreatment

The bovine striatal dopamine D₁ receptor was solubilized with a combination of sodium cholate and NaCl in the presence of phospholipids, following treatment of membranes with a dopaminergic agonist (SKF-82526-J) or antagonist (SCH-23390). The solubilized receptors were subsequently reconstituted into lipid vesicles by gel-filtration. A comparison of ligand-binding properties shows that the solubilized and reconstituted receptors bound [³H]SCH-23390 to a homogeneous site in a saturable, stereospecific and reversible manner with a K_d of 0.95 and 1.1 nM and a B_max of 918 and 885 fmol/mg protein respectively for agonist- and antagonist-pretreated preparations. These values are very similar to those obtained for membrane-bound receptors. The competition of antagonists for [³H]SCH-23390 binding exhibited a clear D₁ dopaminergic order in the reconstituted preparation obtained from either agonist or antagonist-pretreated membranes, except that (+)butaclamol was about four-fold more potent thancis-flupentixol in displacing [³H]SCH-23390 binding in preparation obtained from agonist-pretreated membranes compared to antagonist-pretreated membranes. The agonist/[³H]SCH-23390 competition studies revealed the presence of a highaffinity component of agonist binding in both the reconstituted receptor preparations. The number of high-affinity agonist binding sites, however, is 40–80% higher in reconstituted preparation obtained from antagonist-treated membrane compared to that obrained from the agonist-treated membrane. In both the preparations, 100 M guanylylimidodiphosphate (Gpp(NH)p) completely abolished the high-affinity component of agonist binding compared to partial abolition in the native membranes, indicating a close association of a G-protein with the solubilized receptors. Whether the receptor was solubilized following agonist or antagonist preincubation of the membranes, the receptor-detergent complex eluted from a steric-exclusion HPLC column with an apparent molecular size of 360,000. Preincubation of the solubilized preparations with Gpp(NH)p had virtually no effect on the elution profile suggesting a lack of guanine nucleotide-dependent dissociation of G-protein receptor complex.     

Evidence for specific binding sites for guanine nucleotides in adipocyte and hepatocyte plasma membranes. A difference in fate of GTP and guanosine 5'-(beta, gamma-imino) triphosphate.

Binding and degradation of GTP and guanosine 5'-(beta, gamma-imino)triphosphate (Gpp(NH)p by plasma membranes from rat liver and fat cells were investigated. Gpp(NH)p is hydrolyzed predominantly by nucleotide pyrophosphohydrolases in the membranes, whereas GTP is hydrolyzed primarily by nucleotide phosphohydrolases. These enzymes are not specific for the guanine nucleotides since co-addition of the analogous adenine nucleotides spares their hydrolysis. Both Gpp(NH)p and GTP are taken up by the membranes at sites which, to the extent that high concentrations of the corresponding adenine nucleotides fail to inhibit uptake, appear to be specific for guanine nucleotides. Gpp(NH)p taken up at these sites remains essentially intact irrespective of the degree of hydrolysis of unbound Gpp(NH)p by nucleotide pyrophosphohydrolases, indicating that the binding siteis incapable of degrading Gpp(NH)p. GTP and GDP inhibit competitively the binding of Gpp(NH)p; the binding constants for the three nucleotides are similar (0.1 to 0.4 muM) and are in the same range required for their effects on adenylate cyclase activity. Binding of the nucleotides is inhibited by sulfhydryl agents, suggesting that a sulfhydryl group is involved in the binding process. In contrast to binding of Gpp(NH)p, uptake of GTP is accompanied by substantial hydrolysis, primarily to GDP, under incubation conditions (high [ATP] plus ATP regenerating system) in which [GTP] in the medium remains essentially constant. GDP bound to the membranes is progressively hydrolyzed to 5'-GMP. Thus, GTP and Gpp(NH)p, although binding to the same specific sites, are differentially susceptible to hydrolysis at their terminal phosphates when bound to these sites. These findings are discussed in terms of the markedly different potencies of GTP and Gpp(NH)p as activators of adenylate cyclase systems.