The positive charge at Lys-288 of the glucagon-like peptide-1 (GLP-1) receptor is important for binding the N-terminus of peptide agonists

Lysine-288 in the glucagon-like peptide-1 receptor was predicted to be ideally positioned to play a role in hormone binding. Subsequent mutation of Lys-288 to Ala or Leu greatly reduced hormone affinity, while substitution with Arg had minimal effect. Compared to wild type, the Lys288-Ala receptor had a reduced affinity for three peptide ligands with complete N-terminal sequences but not for their N-truncated analogues. Hence, the role of this positively charged residue, which is conserved at the equivalent position in all other Family B receptors, was determined to be important for receptor interaction with the N-terminal eight residues of peptide agonists.     

ASP147 in the third transmembrane helix of the rat mu opioid receptor forms ion-pairing with morphine and naltrexone.

We tested the hypotheses that the carboxylate side chain of Asp147 of the mu opioid receptor interacts with the protonated nitrogen of naltrexone and morphine and that this interaction is important for pharmacological properties of the two compounds. Mutation of Asp147 to Ala or Asn substantially reduced the affinity of naltrexone and the affinity, potency and efficacy of morphine, while the Glu mutant had similar properties as the wildtype, indicating the significant role of the carboxylate group of Asp147 in receptor binding and activation. This role could be due to its direct interaction with ligands or involvement in interhelical interactions. The unprotonated analogs of naltrexone and morphine, cyclopropylcarbonyl noroxymorphone (CPCNOM) and N-formylnormorphine (NFNM), respectively, were used to discriminate between these mechanisms. CPCNOM was much less potent as an antagonist and had substantially lower affinity for the mu receptor than naltrexone. Similarly, NFNM was unable to activate the mu receptor and had much lower affinity than morphine. These results indicate the importance of the protonated nitrogen. Notably, the D147A and D147N mutations did not appreciably affect the binding affinities of CPCNOM and NFNM. In addition, the D147E mutant had similar affinities for CPCNOM and NFNM as the D147A and D147N mu receptors. Thus, the carboxylate group of Asp147 is not important for binding of the two unprotonated compounds. These results indicate that the carboxylate group of Asp147 of the mu receptor interacts directly with the protonated nitrogen of naltrexone and morphine and this interaction is important for binding and receptor activation.     

Rat brain TRH receptors: kinetics, pharmacology, distribution and ionic effects

Optimal conditions for measuring receptor binding for thyrotropin-releasing hormone (TRH) in the rat central nervous system (CNS) have been determined using 3H-labelled [3-Me-His2]TRH [( 3H]MeTRH). Binding assays conducted at 0 degree C for 5-6 h using sodium phosphate- and/or Hepes-buffered tissue resuspensions, with subsequent filtration through Whatman GF/B filters, yielded the best results. Association and dissociation of [3H]MeTRH binding to amygdala membranes were time and temperature dependent. Dissociation kinetics appeared biphasic. Progressive reduction in receptor affinity and capacity and increased radioligand breakdown were observed at elevated temperatures. Bacitracin (25-1000 microM) prevented peptide degradation but inhibited receptor binding (8-37%). Detailed competition experiments using MeTRH and other drugs yielded a pharmacological profile similar to that observed previously in other tissues indicating TRH receptor identification. Highest density of TRH receptors was observed in the retina and numerous limbic areas. Monovalent and divalent cations modulated [3H]MeTRH binding by reducing apparent receptor number.     

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.     

Pituitary thyrotropin-releasing hormone (TRH) receptors: effects of TRH, drugs mimicking TRH action, and chlordiazepoxide

Binding of TRH to specific cell surface receptors on clonal GH4C1 cells is followed within 10 min by receptor sequestration and over 24 h by receptor down-regulation. These experiments were designed to determine if TRH-activated second messenger systems are responsible for changes in receptor localization or number. BAY K8644 and A23187, which increase intracellular calcium, alone or together with 12-O-tetradecanoyl phorbol acetate (TPA), which activates protein kinase C, did not appear to internalize TRH receptors. Drug treatment did not alter the rate of [3H]MeTRH association or internalization, determined by resistance to an acid/salt wash, or the amount of [3H]MeTRH able to bind at 0 C, where only surface receptors are accessible. TPA (0-100 nM) alone or in combination with BAY K8644 or A23187, also failed to change receptor number or affinity after 48 h when TRH caused a 75% decrease in the density of specific binding sites. Chlordiazepoxide has been reported antagonize TRH binding and TRH-induced phospholipid breakdown. Chlordiazepoxide shifted the dose-response curves for TRH stimulation of PRL release and synthesis to the right, and did not change PRL release alone. The affinity of receptors for chlordiazepoxide was not affected by a nonhydrolyzable analog of GTP whereas affinity for TRH was decreased; these properties are consistent with the classification of chlordiazepoxide as a competitive antagonist. Several experiments tested whether chlordiazepoxide would cause receptor internalization and down-regulation. Chlordiazepoxide did not appear to internalize TRH receptors, because TRH-binding sites became available rapidly and at the same rate after they had been saturated with chlordiazepoxide at 0 or 37 C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible role for ligand binding of histidine 81 in the second transmembrane domain of the rat prostaglandin F2alpha receptor

For the five principal prostanoids PGD2, PGE2, PGF2alpha, prostacyclin and thromboxane A2 eight receptors have been identified that belong to the family of G-protein-coupled receptors. They display an overall homology of merely 30%. However, single amino acids in the transmembrane domains such as an Arg in the seventh transmembrane domain are highly conserved. This Arg has been identified as part of the ligand binding pocket. It interacts with the carboxyl group of the prostanoid. The aim of the current study was to analyze the potential role in ligand binding of His-81 in the second transmembrane domain of the rat PGF2alpha receptor, which is conserved among all PGF2alpha receptors from different species. Molecular modeling suggested that this residue is located in close proximity to the ligand binding pocket Arg 291 in the 7th transmembrane domain. The His81 (H) was exchanged by site-directed mutagenesis to Gln (Q), Asp (D), Arg (R), Ala (A) and Gly (G). The receptor molecules were N-terminally extended by a Flag epitope for immunological detection. All mutant proteins were expressed at levels between 50% and 80% of the wild type construct. The H81Q and H81D receptor bound PGF2alpha with 2-fold and 25-fold lower affinity, respectively, than the wild type receptor. Membranes of cells expressing the H81R, H81A or H81G mutants did not bind significant amounts of PGF2alpha. Wild type receptor and H81Q showed a shallow pH optimum for PGF2alpha binding around pH 5.5 with almost no reduction of binding at higher pH. In contrast the H81D mutant bound PGF2alpha with a sharp optimum at pH 4.5, a pH at which the Asp side chain is partially undissociated and may serve as a hydrogen bond donor as do His and Gln at higher pH values. The data indicate that the His-81 in the second transmembrane domain of the PGF2alpha receptor in concert with Arg-291 in the seventh transmembrane domain may be involved in ligand binding, most likely not by ionic interaction with the prostaglandin's carboxyl group but rather as a hydrogen bond donor.     

A novel substance P binding site in rat brain regions modulates TRH receptor binding

Binding sites for thyrotropin-releasing hormone (TRH) were labelled with [³H](2-Me-His³)TRH ([³H]MeTRH) on membranes from rat brain regions at 0°C for 5 h. Amygdaloid membranes bound [³H]MeTRH with high-affinity (K _d=3.1±0.5 nM (n=4)). Five TRH analogs competed for this binding with the same rank order and with affinities that matched the pharmacological specificity of pituitary TRH receptors. Substance P (SP) and its C-terminal fragments reduced amygdaloid TRH receptor binding in a concentration dependent manner (IC₅₀ for SP=65 M). The rank order of potency of SP analogs at inhibiting TRH receptor binding was: SP>nonapeptide (3–11)>hexapeptide (6–11)>heptapeptide (5–11)>pentapeptide (7–11). However, other tachykinins were inactive in this system. SP was a potent inhibitor of [³H]MeTRH binding in hippocampus> spinal cord>retina>n. accumbens>hypothalamus>amygdaloid>olfactory bulb pituitary>pons/medulla in parallel assays. In amygdaloid membranes SP (50 M) reduced the apparent maximum receptor density by 39% (p<0.01) without altering the binding affinity, and 100 M SP induced a biphasic dissociation of [³H]MeTRH with kinetics faster than those induced by both TRH (10 M) and serotonin (100 M). In contrast, other neuropeptides such as neurotensin, proctolin, angiotensin II, bombesin and luteinizing hormone releasing hormone did not significantly inhibit [³H]MeTRH binding to amydaloid membranes. Thus, the SP site with low affinity in the rat brain is not like any of the previously described tachykinin/neurokinin binding sites but resembles the site found on neuroblastoma cells (108CC15) and on adrenal chromaffin cells that modulate cation permeability and nicotinic receptors respectively. The physiological role of these atypical SP sites in the rat brain remains to be determined.A preliminary account of these studies has been presented to the British Pharmacological Society (9).     

Evidence for a direct interaction between the penultimate aspartic acid of cholecystokinin and histidine 207, located in the second extracellular loop of the cholecystokinin B receptor.

Recently, we reported that the mutation of His(207) to Phe located in the second extracellular loop of the cholecystokinin B receptor strongly affected cholecystokinin (CCK) binding (Silvente-Poirot, S., Escrieut, C., and Wank, S. A. (1998) Mol. Pharmacol. 54, 364-371). To characterize the functional group in CCK that interacts with His(207), we first substituted His(207) to Ala. This mutation decreased the affinity and the potency of CCK to produce total inositol phosphates 302-fold and 456-fold without affecting the expression of the mutant receptor. The screening of L-alanine-modified CCK peptides to bind and activate the wild type and mutant receptors allowed the identification of the interaction of the C-terminal Asp(8) of CCK with His(207). The H207A-CCKBR mutant, unlike the wild type receptor, was insensitive to substitution of Asp(8) of CCK to other amino acid residues. This interaction was further confirmed by mutating His(207) to Asp. The affinity of CCK for the H207D-CCKBR mutant was 100-fold lower than for the H207A-CCKBR mutant, consistent with an electrostatic repulsion between the negative charges of the two interacting aspartic acids. Peptides with neutral amino acids in position eight of CCK reversed this effect and displayed a gain of affinity for the H207D mutant compared with CCK. To date, this is the first report concerning the identification of a direct contact point between the CCKB receptor and CCK.     

Residue 21 of human granulocyte-macrophage colony-stimulating factor is critical for biological activity and for high but not low affinity binding.

The functional role of the predicted first alpha-helix of human granulocyte-macrophage colony-stimulating factor (GM-CSF) was analysed by site-directed mutagenesis and multiple biological and receptor binding assays. Initial deletion mutagenesis pointed to residues 20 and 21 being critical. Substitution mutagenesis showed that by altering Gln20 to Ala full GM-CSF activity was retained but that by altering Glu21 for Ala GM-CSF activity and high affinity receptor binding were decreased. Substitution of different amino acids for Glu21 showed that there was a hierarchy in the ability to stimulate the various biological activities of GM-CSF with the order of potency being Asp21 greater than Ser21 greater than Ala21 greater than Gln21 greater than Lys21 = Arg21. To distinguish whether position 21 was important for GM-CSF binding to high or low affinity receptors, GM-CSF (Arg21) was used as a competitor for [125I]GM-CSF binding to monocytes that express both types of receptor. GM-CSF (Arg21) exhibited a greatly reduced capacity to compete for binding to high affinity receptors, however, it competed fully for [125I]GM-CSF binding to low affinity receptors. Furthermore, GM-CSF (Arg21) was equipotent with wild-type GM-CSF in binding to the cloned low affinity alpha-chain of the GM-CSF receptor. These results show that (i) this position is critical for high affinity but not for low affinity GM-CSF receptor binding thus defining two functional parts of the GM-CSF molecule; (ii) position 21 of GM-CSF is critical for multiple functions of GM-CSF; and (iii) stimulation of proliferation and mature cell function by GM-CSF are mediated through high affinity receptors.     

Europium-labeled melanin-concentrating hormone analogues: ligands for measuring binding to melanin-concentrating hormone receptors 1 and 2

We investigated the use of Eu3+ chelate-labeled analogues of melanin-concentrating hormone (MCH) as ligands for both human MCH receptors (MCHR1 and MCHR2). The analogues employed were Ala17 MCH, S36057 (Y-ADO-RC*MLGRVFRPC*W, where ADO=8-amino-3,6-dioxyoctanoyl and *=disulfide bond), and R2P (RC*MLGRVFRPC*Y-NH2). The peptides were readily labeled on the alpha-amino residue with the Eu3+ chelate of N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3-tetraacetic acid and then purified by reverse-phase fast-performance liquid chromatography at neutral pH to maintain Eu3+ chelation. Both labeled Ala17 MCH and S36057 had high affinity for MCHR1 ( Kd = 0.37 and 0.059nM, respectively) while Eu3+ -labeled S36057 and R2P had high affinity for MCHR2 ( Kd = 0.16 and 0.10nM, respectively). Labeled Ala17 MCH had little demonstrable binding affinity for MCHR2. Eu3+ -labeled S36057 and R2P were full agonists at MCHR1 when assessed by measurement of agonist-stimulated GTPgamma(35)S binding. Competition binding experiments with both MCHR isoforms, a series of previously characterized alanine scan MCH analogues, and a recently identified nonpeptide MCHR1-selective antagonist T-226296 confirmed the expected receptor selectivity. These studies further extend the utility of Eu3+ chelate time-resolved fluorescence for the development of high-sensitivity, nonradioactive receptor binding assays and demonstrate the need to select the optimal ligand for labeling.     

Lack of Usefulness of DN-1417 for Characterization of a CNS Receptor for Thyrotropin-Releasing Hormone

CNS receptors for thyrotropin-releasing hormone (TRH) and its analogs are likely to mediate the experimentally and clinically observed net excitatory effect of these peptides on lower motor neurons. Previous findings suggest that several types of TRH receptors with distinct TRH analog specificities may be present in rat CNS. In particular, based on competition isotherm assays with unlabeled analog gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolineamide (DN-1417). Funatsu et al. claim the existence of a limbic forebrain site that binds this peptide and TRH with high affinity but that does not bind [3-methyl-histidyl2]-TRH (MeTRH). Using saturation and competition isotherm experiments, we have examined the binding of [3H]TRH and [3H]DN-1417 in three regions of rat CNS: pyriform cortex/amygdala, limbic forebrain, and lumbosacral spinal cord. In all three regions, saturation assays with [3H]TRH (0.4-100 nM) resolved only a single, saturable receptor with high affinity (KD = 12-14 nM) for TRH; in no case could more than one saturable site be identified. When [3H]DN-1417 was substituted as the assay ligand, no high-affinity binding component for this analog could be detected in the three regions. Competition curves for the binding of unlabeled DN-1417 to limbic forebrain and lumbosacral spinal cord ([3H]TRH as assay ligand) were monophasic (not biphasic like those of Funatsu et al.) and indicative of low-affinity binding of DN-1417 in these regions (Ki values = 2-3 microM; in agreement with values obtained in similar assays with [3H]MeTRH).(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of Arg182 in the second extracellular loop of angiotensin II receptor AT2 in ligand binding.

The phenolic side chain of Tyr(4) present in Ang II is proposed to interact with the side chain of Arg 167 of the AT1 receptor. To determine the contribution of the analogous Arg182 in the ligand-binding properties of the AT2, we replaced the Arg182 with Glu and Ala, and analyzed the ligand-binding properties. Our results suggest that replacing Arg182 with either Glu or Ala abolished the ability of the AT2 receptor to bind the nonspecific peptidic ligands, (125)I-Ang II and [(125)I-Sar(1)-Ile(8)]Ang II, as well as the AT2 receptor-specific peptidic ligand (125)I-CGP42112A. We have shown previously that replacing the positively charged side chain of Lys215 with the negatively charged side chain of Glu in the fifth TMD did not alter the high affinity binding of (125)I-CGP42112A to the AT2 receptor. However, ligand-binding properties of the Arg182Glu mutant suggest that positively charged side chain of Arg182 located in the junction of second ECL and the fourth TMD is critical for high affinity binding of all three peptidic ligands to the AT2 receptor.     

Hemoglobin providence. Functional consequences of two alterations of the 2,3-diphosphoglycerate binding site at position beta 82.

Position beta 82 in human hemoglobin (Hb) is normally occupied by lysine, a positively charged residue that is involved in the binding of anionic cofactors. This residue is substituted by a neutral residue in Hb Providence Asn and by a negatively charged residue in Hb Providence Asp. Hb Providence Asp shows more differences from Hb A than does Hb Providence Asn in studies of the kinetics and equilibria of ligand binding. For both forms, homotropic (cooperative) interactions are normal with n values of 2.5 to 2.7, while heterotropic (pH and anion) interactions are reduced greatly. The reduction in anion sensitivity is attributed to the absence of a positive residue at position beta 82. Reduction in pH sensitivity may be due to a ligand-linked change in the pK of a neighboring residue, beta 143 histidine, which normally is not a Bohr group. This change in pK would act in opposition to the normal Bohr effect. Reduction in the net positive charge of the central cavity has a further consequence. Relative to Hb A, both Hb Providence Asn and Hb Providence Asp show decreased oxygen affinities at neutral pH in the absence of cofactors. This suggests that in Hb A the binding of anionic cofactors directly influences the oxygen affinity by neutralizing the charged groups of the diphosphoglycerate binding site and thus stabilizing the low affinity (T) conformation. From pH 6 to 9 in the presence of 1 M NaCl, where all the charged groups may be masked, the oxygen-binding properties of Hb A and the Hb Providence mutants are identical. Moreover, subunit dissociation of the liganded Hb Providence mutants appears to be increased, as is known to occur for Hb A in the presence of high salt. The results obtained with Hb Providence Asn and Hb Providence Asp illustrate how single amino acid substitutions can modify hemoglobins' pH and anion interactions without altering cooperative interactions between subunits. The alteration in cofactor effects observed with these mutants also illustrates differences between the allosteric effects induced by organic and inorganic anions.     

Proliferation of thyrotropin releasing hormone receptors in specific brain regions during the development of hypertension in spontaneously hypertensive rats

The binding of [3H] [3-MeHis2] thyrotropin releasing hormone [( 3H]MeTRH) to brain membranes prepared from 8 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. [3H]MeTRH bound specifically to rat brain membranes at a single high affinity site. The density (Bmax value) of [3H]MeTRH binding sites was significantly greater (28%) in SHR rats compared to WKY rats. The apparent dissociation constants (Kd values) for the binding of [3H]MeTRH in SHR and WKY rats did not differ. Binding in the various brain regions revealed that the density of [3H]MeTRH was highest in the hypothalamus followed in decreasing order by pons + medulla, midbrain, cortex and striatum. The binding of [3H]MeTRH was approximately 25% greater in cortex, hypothalamus and striatum of SHR rats in comparison to WKY rats. The binding in pons + medulla, midbrain and pituitary of SHR and WKY rats did not differ. To assess the significance of increased binding sites for [3H]MeTRH in some brain regions of SHR rats, the binding studies were carried out during normotensive and hypertensive stages of postnatal age in the two strains. In 3 and 4 week old SHR rats there was neither an increase in blood pressure nor any increase in [3H]MeTRH binding in the hypothalamus and striatum as compared to age matched WKY rats. With the development of elevated blood pressure at 6 weeks, an increase in [3H]MeTRH binding in the hypothalamus and striatum of SHR rats in comparison to the tissues from WKY rats was observed. The results provide, for the first time, evidence for a parallel increase in the density of brain TRH receptors with elevation of blood pressure, and suggest that brain TRH receptors may play an important role in the pathophysiology of hypertension.     

Sulfhydryl Groups in Receptor Binding of Thyrotropin-Releasing Hormone to Rat Amygdala

Abstract: Binding of [³H]-[3-Me-His²]thyrotropin-releasing hormone ([³H]MeTRH) to TRH receptors in rat amygdala was decreased by sulfhydryl reagents in a time-, temperature-, and concentration-dependent manner. A pronounced reduction in receptor density, with little or no change in binding affinity, was apparent following disulfide bond reduction by dithiothreitol (DTT), alkylation of thiol groups by N -ethylmaleimide (NEM), and their oxidation by 5,5'-dithiobis (2-nitrobenzoic acid). Heavy metals (Cd²⁺, Hg²⁺), which complex with reactive -SH residues, also potently inhibited binding. The pharmacological specificity of residual [³H]MeTRH binding in chemically modified amygdala membranes was the same as that in control preparations. Sequential exposure to thiol reagents, in the presence or absence of cations, revealed possible additive effects. Pretreatment of membranes with TRH (10^--⁸-^-10^--⁶ M ), and its continued presence during modification, afforded protection against DTT and NEM. These results indicate the possible importance of thiol groups in the maintenance of TRH receptor conformation.     

Modulation of Receptors for Thyrotropin-Releasing Hormone by Benzodiazepines: Brain Regional Differences

The benzodiazepines (BZDs) chlordiazepoxide (CDE), diazepam (DZM), and flurazepam (FLM) inhibited receptor binding for thyrotropin-releasing hormone (TRH) with low micromolar potency. In contrast, numerous other categories of drugs were previously shown to be inactive. Scatchard analysis of competition data suggested that the BZDs reduced TRH receptor affinity, consistent with competitive inhibition. Receptors from amygdala, retina, and pituitary appeared more sensitive to inhibition by BZDs than those from hypothalamus, hippocampus, spinal cord, or cerebellum. The latter four regions also gave shallower inhibition curves. CDE revealed an apparently biphasic dissociation of [3-Me-His2]TRH([3H]MeTRH) from amygdala membranes at 4 degrees C, with kinetics similar to those with TRH. These results suggest that TRH receptors in the brain are heterogeneous and that certain BZDs in high therapeutic concentrations may exert central effects through actions at TRH receptors or coupled proteins.     

Protein kinase A-mediated phosphorylation of the beta 2-adrenergic receptor regulates its coupling to Gs and Gi. Demonstration in a reconstituted system

While classically viewed as a prototypic G(s) and adenylyl cyclase-coupled G protein-coupled receptor, recent studies have indicated that some aspects of beta(2)-adrenergic receptor (beta(2)-AR) signaling are inhibited by pertussis toxin, indicating that they are mediated by G(i)/G(o) proteins. These signals include activation of ERK MAPKs and Akt activation, as well as hypertrophic and anti-apoptotic pathways in cardiac myocytes. Studies in cultured cells have suggested the hypothesis that protein kinase A (PKA)-mediated phosphorylation of the beta(2)-AR regulates its coupling specificity with respect to G(s) and G(i). Using a Chinese hamster ovary cell system, we show that mutant beta(2)-ARs with Ala substituted for Ser at consensus PKA sites stimulate robust cyclic AMP accumulation (G(s)) but are unable to activate ERK (G(i)). In contrast, Ser --> Asp mutants are dramatically impaired in their ability to activate adenylyl cyclase but are significantly more active than wild type receptor in activating ERK. Activation of adenylyl cyclase by wild type and Ser --> Ala mutant receptors is not altered by pertussis toxin, whereas adenylyl cyclase stimulated through the Ser --> Asp mutant is enhanced. Activation of ERK by wild type and Ser --> Asp receptors is inhibited by pertussis toxin. To further rigorously test the hypothesis, we utilized a completely reconstituted system of purified recombinant wild type and PKA phosphorylation site mutant beta(2)-ARs and heterotrimeric G(s) and G(i). G protein coupling was measured by receptor-mediated stimulation of GTPgammaS binding to the G protein. PKA-mediated phosphorylation of the beta(2)-AR significantly decreased its ability to couple to G(s), while simultaneously dramatically increasing its ability to couple to G(i). These results are reproduced when a purified recombinant Ser --> Asp mutant beta(2)-AR is tested, whereas the Ser --> Ala receptor resembles the unphosphorylated wild type. These results provide strong experimental support for the idea that PKA-mediated phosphorylation of the beta(2)-adrenergic receptor switches its predominant coupling from G(s) to G(i).     

Agonist occupancy of a single monomeric element is sufficient to cause internalization of the dimeric beta2-adrenoceptor

A range of studies have indicated that many rhodopsin-like, family A G protein-coupled receptors, including the beta(2)-adrenoceptor, exist and probably function as dimers. It is less clear if receptors internalize as dimers and if agonist occupancy of only one element of a dimer is sufficient to cause internalization of a receptor dimer into the cell. We have used a chemogenomic approach to demonstrate that this is the case. Following expression of the wild type beta(2)-adrenoceptor, isoprenaline but not 1-(3'4'-dihydroxyphenyl)-3-methyl-1-butanone, which does not have significant affinity for the wild type receptor, caused receptor internalization. By contrast, 1-(3'4'-dihydroxyphenyl)-3-methyl-1-butanone, but not isoprenaline that does not have high affinity for the mutated receptor, caused internalization of Asp(113)Serbeta(2)-adrenoceptor. Following co-expression of wild type and Asp(113)Serbeta(2)-adrenoceptors each of isoprenaline and 1-(3'4'-dihydroxyphenyl)-3-methyl-1-butanone caused the co-internalization of both of these two forms of the receptor. Co-expressed wild type and Asp(113)Serbeta(2)-adrenoceptors were able to be co-immunoprecipitated and 1-(3'4'-dihydroxyphenyl)-3-methyl-1-butanone produced internalization of the wild type receptor that was not prevented by the beta-adrenoceptor antagonist propranolol that binds with high affinity only to the wild type receptor. These results demonstrate that agonist occupancy of either single binding site of the beta(2)-adrenoceptor dimer is sufficient to cause internalization of the dimer and that antagonist occupation of one of the two ligand binding sites is unable to prevent agonist-mediated internalization.     

Ligand binding studies of engineered cytochrome P-450d wild type, proximal mutants, and distal mutants

Interactions of various axial ligands with cytochrome P-450d wild type, proximal mutants (Lys453Glu, Ile460Ser), and putative distal mutants (Glu318Asp, Thr319Ala, Thr322Ala) expressed in yeast were studied with optical absorption spectroscopy. P-450d wild type and all five mutants were purified essentially as the high-spin form, but the putative distal mutants contained about 5% low-spin form. Bindings of metyrapone and 4-phenylimidazole to the wild type and all mutants formed nitrogen-bound low-spin forms. In contrast, binding of 2-phenylimidazole to the wild type and most of mutants formed oxygen-bond low-spin forms except for the mutant Glu318Asp in which the nitrogen-bound low-spin form was formed. By analogy with the distal structure of P-450cam, it was thus suggested that Glu318 of P-450d, which corresponds with Asp251 of P-450cam, somehow interacts with 2-phenylimidazole over the heme plane. Addition of 1-butanol and acetanilide, a substrate of P-450d, to the wild type and mutants caused the spin change to the low-spin form. The order of dissociation constants of these oxygen ligands to P-450d was wild type greater than proximal mutants greater than putative distal mutants. Spectral analyses showed that the binding of acetanilide is the same as that of another substrate, 7-ethoxycoumarin, in the putative distal mutants but is not the same in the wild type and proximal mutants. From these findings together with other spectral data, it was suggested that the region from Glu318 to Thr322 is located at the distal region of the heme in membrane-bound P-450d as suggested from the X-ray crystal structure of water-soluble P-450cam and amino acid alignments of P-450s.     

Dermenkephalin and deltorphin I reveal similarities within ligand-binding domains of mu- and delta-opioid receptors and an additional address subsite on the delta-receptor.

Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) are the first naturally occurring peptides highly potent for and almost specific to the mu- and delta-opioid receptors, respectively. The amino-terminal domains Tyr-D-X-Phe (where X is either Ala or Met) of these peptides behave as selective and potent mu-receptor ligands. Routing of Tyr-D-X-Phe to the delta- or the mu- receptor is associated with the presence or the absence at the C-terminus of an additional hydrophobic and negatively charged tetrapeptide by-passing the mu-addressing ability of the amino-terminal moiety. A study of 20 Tyr-D-X-Phe-Y-NH2 analogs with substitution of X and Y by neutral, hydrophobic, aromatic amino acids as well as by charged amino acid residues shows that tetrapeptides maintain high binding affinity and selectivity for the mu-opioid receptor. Although residue in position 4 serves a delta-address function, the tripeptide motif at the C-terminus of dermenkephalin and deltorphin I are critical components for high selectivity at delta-opioid receptor. Results demonstrate that mu- and delta-opioid receptors share topologically equivalent ligand-binding domains, or ligand-binding sequences similarities, that recognized Tyr-D-X-Phe as a consensus message-binding sequence. The delta-receptor additionally contains a unique address subsite at or near the conserved binding domain that accommodates the C-terminal tetrapeptide motif of dermenkephalin and deltorphin I.