Conserved aromatic residues in the transmembrane region VI of the V1a vasopressin receptor differentiate agonist vs. antagonist ligand binding.

Despite their opposite effects on signal transduction, the nonapeptide hormone arginine-vasopressin (AVP) and its V1a receptor-selective cyclic peptide antagonist d(CH2)5[Tyr(Me)2]AVP display homologous primary structures, differing only at residues 1 and 2. These structural similarities led us to hypothesize that both ligands could interact with the same binding pocket in the V1a receptor. To determine receptor residues responsible for discriminating binding of agonist and antagonist ligands, we performed site-directed mutagenesis of conserved aromatic and hydrophilic residues as well as nonconserved residues, all located in the transmembrane binding pocket of the V1a receptor. Mutation of aromatic residues of transmembrane region VI (W304, F307, F308) reduced affinity for the d(CH2)5[Tyr(Me)2]AVP and markedly decreased affinity for the unrelated strongly hydrophobic V1a-selective nonpeptide antagonist SR 49059. Replacement of these aromatic residues had no effect on AVP binding, but increased AVP-induced coupling efficacy of the receptor for its G protein. Mutating hydrophilic residues Q108, K128 and Q185 in transmembrane regions II, III and IV, respectively, led to a decrease in affinity for both agonists and antagonists. Finally, the nonconserved residues T333 and A334 in transmembrane region VII, controlled the V1a/V2 binding selectivity for both nonpeptide and cyclic peptide antagonists. Thus, because conserved aromatic residues of the V1a receptor binding pocket seem essential for antagonists and do not contribute at all to the binding of agonists, we propose that these residues differentiate agonist vs. antagonist ligand binding.     

Arginine and cysteine residues in the ligand binding site of alpha 2-adrenergic receptors

alpha 2-Adrenergic receptors were identified in calf brain, human platelet and human uterus membranes by [3H]-rauwolscine binding. The reagents phenylglyoxal (selective for guanidino groups), p- hydroxy mercuribenzoate and N-ethylmaleimide (selective for sulfhydryl groups) caused a time- and dose- dependent decrease in the number of receptor sites. alpha 2-Adrenergic agonists and antagonists mediated efficient protection of the receptors against these reagents. These data suggest that essential arginine and cysteine residues are present at or near the alpha 2-adrenergic binding site.     

Interaction between clozapine and a lipophilic alpha 1-adrenergic agonist

Acute intraperitoneal injection of clozapine produced marked hypothermia and ataxia in Swiss-Webster mice. These two effects were almost completely blocked by the lipophilic alpha 1-adrenergic agonist, St 587, but not by the peripherally-acting alpha 1 agonist methoxamine. It was inferred that these effects of clozapine are central in origin and probably resulted from alpha 1 adrenergic blockade. However, since prazosin, a selective alpha 1-adrenergic antagonist did not elicit either hypothermia or ataxia in mice it became clear that the alpha 1 adrenergic blocking effect of clozapine is not entirely responsible for these effects, but has a major contributory role in their production. Both clozapine and prazosin inhibited the d-amphetamine-induced locomotor stimulation in mice. St 587 did not significantly reduce this amphetamine-blocking effect of clozapine. It was inferred that this response to d-amphetamine involving the release of mesolimbic dopamine is distinct from the other two St 587-sensitive responses. The hypothermic and ataxic effects of clozapine developed complete tolerance after just four days of treatment, but ten days of such treatment was required for the development of tolerance to the amphetamine-blocking effect of clozapine. The possible relationships between St 587-sensitive and insensitive effects of clozapine and its antipsychotic property are discussed.     

Roles for loop 2 residues of alpha1 glycine receptors in agonist activation

The present study tested the hypothesis that several residues in Loop 2 of alpha1 glycine receptors (GlyRs) play important roles in mediating the transduction of agonist activation to channel gating. This was accomplished by investigating the effect of cysteine point mutations at positions 50-60 on glycine responses in alpha1GlyRs using two-electrode voltage clamp of Xenopus oocytes. Cysteine substitutions produced position-specific changes in glycine sensitivity that were consistent with a beta-turn structure of Loop 2, with odd-numbered residues in the beta-turn interacting with other agonist-activation elements at the interface between extracellular and transmembrane domains. We also tested the hypothesis that the charge at position 53 is important for agonist activation by measuring the glycine response of wild type (WT) and E53C GlyRs exposed to methanethiosulfonate reagents. As earlier, E53C GlyRs have a significantly higher EC(50) than WT GlyRs. Exposing E53C GlyRs to the negatively charged 2-sulfonatoethyl methanethiosulfonate, but not neutral 2-hydroxyethyl methanethiosulfonate, positively charged 2-aminoethyl methanethiosulfonate, or 2-trimethylammonioethyl methanethiosulfonate, decreased the glycine EC(50) to resemble WT GlyR responses. Exposure to these reagents did not significantly alter the glycine EC(50) for WT GlyRs. The latter findings suggest that the negative charge at position 53 is important for activation of GlyRs through its interaction with positive charge(s) in other neighboring agonist activation elements. Collectively, the findings provide the basis for a refined molecular model of alpha1GlyRs based on the recent x-ray structure of a prokaryotic pentameric ligand-gated ion channel and offer insight into the structure-function relationships in GlyRs and possibly other ligand-gated ion channels.     

Norepinephrine- and phorbol ester-induced phosphorylation of alpha(1a)-adrenergic receptors. Functional aspects

Maximal adrenergic responses in Rat-1 fibroblasts expressing alpha(1a)-adrenergic receptors are not blocked by activation of protein kinase C. In contrast, activation of protein kinase C induces the phosphorylation of alpha(1b)-adrenoreceptors and blocks their actions. The effect of norepinephrine and phorbol esters on alpha(1a)-adrenoreceptor phosphorylation and coupling to G proteins were studied. Both stimuli lead to dose-dependent receptor phosphorylation. Interestingly, protein kinase C activation affected to a much lesser extent the actions of alpha(1a)-adrenergic receptors than those of the alpha(1b) subtype (norepinephrine elicited increases in calcium in whole cells and [(35)S]GTPgammaS binding to membranes). Basal phosphorylation of alpha(1a)-adrenergic receptors was much less than that observed with the alpha(1b) subtype. The carboxyl terminus seems to be the main domain for receptor phosphorylation. Therefore, chimeric receptors, where the carboxyl-terminal tails of alpha(1a) and alpha(1b) adrenergic receptors were exchanged, were constructed and expressed. alpha(1a)-Adrenoreceptors wearing the carboxyl tail of the alpha(1b) subtype had a high basal phosphorylation and displayed a strong phosphorylation in response to norepinephrine and phorbol esters. Our results demonstrate that stimulation of alpha(1a)-adrenergic receptor, or activation of protein kinase C, leads to alpha(1a)-adrenergic receptor phosphorylation. alpha(1a)-Adrenoreceptors are affected to a much lesser extent than alpha(1b)-adrenoreceptors by protein kinase C activation.     

Phorbol esters inhibit alpha 1-adrenergic effects and decrease the affinity of liver cell alpha 1-adrenergic receptors for (-)-epinephrine

4 beta-Phorbol 12-myristate 13-acetate (PMA) modified the metabolic actions of three calcium-dependent hormones in different ways. The stimulations of glycogenolysis ureogenesis and phosphatidylinositol labeling produced by alpha 1-adrenergic agonist was blocked by the phorbol ester. In contrast, PMA slightly increased the stimulation of ureogenesis produced by low concentration of angiotensin II without modifying the maximal response. No effect of PMA was observed on the stimulation of ureogenesis induced by vasopressin. The stimulation of phosphatidylinositol labeling induced by vasopressin was decreased by PMA, whereas that induced by angiotensin II was not affected. In intact freshly isolated hepatocytes, [3H]prazosin binds with high affinity to a site which displays the characteristics of alpha 1-adrenergic receptor. Competitive inhibition studies with (-)-epinephrine reveal two different sites for this agonist: a high affinity site (Kd 9 nM) and a low affinity site (Kd 2 microM). In the presence of phorbol esters, (-)-epinephrine binding data now show the presence of a single class of low affinity sites, with similar affinity to those present in control cells. Thus, the inhibition of hepatocyte alpha 1-adrenergic action by PMA may be related to the loss of high affinity binding sites caused by the tumor promoter.     

Identification of residues involved in neurotensin binding and modeling of the agonist binding site in neurotensin receptor 1

The neurotensin receptor 1 (NTR1) subtype belongs to the family of G protein-coupled receptors and mediates most of the known effects of the neuropeptide including modulation of central dopaminergic transmission. This suggested that nonpeptide agonist mimetics acting at the NTR1 might be helpful in the treatment of Parkinson's disease and schizophrenia. Here, we attempted to define the molecular interactions between neurotensin-(8-13), the pharmacophore of neurotensin, and the rat NTR1. Mutagenesis of the NTR1 identified residues that interact with neurotensin. Structure-activity studies with neurotensin-(8-13) analogs identified the peptide residues that interact with the mutated amino acids in the receptor. By taking these data into account, computer-assisted modeling techniques were used to build a tridimensional model of the neurotensin-(8-13)-binding site in which the N-terminal tetrapeptide of neurotensin-(8-13) fits in the third extracellular loop and the C-terminal dipeptide binds to residues at the junction between the extracellular and transmembrane domains of the receptor. Interestingly, the agonist binding site lies on top of the previously described NTR1-binding site for the nonpeptide neurotensin antagonist SR 48692. Our data provide a basis for understanding at the molecular level the agonist and antagonist binding modes and may help design nonpeptide agonist mimetics of the NTR1.     

Impact of aromatic residues within transmembrane helix 6 of the human gonadotropin-releasing hormone receptor upon agonist and antagonist binding.

To investigate the impact of aromatic residues within transmembrane helix 6 (TMH6) of the human gonadotropin-releasing hormone receptor (GnRH-R) on agonist and antagonist binding, residues Y(283), Y(284), W(289), Y(290), W(291), and F(292) were exchanged to alanine and analyzed comprehensively in functional reporter gene and ligand binding assays. Whereas receptor mutants Y(283)A, Y(284)A, and W(291)A were capable of neither ligand binding nor signal transduction, mutants W(289)A, Y(290)A, and F(292)A were functional: the F(292)A mutant behaved like wild-type receptor, while mutants W(289)A and Y(290)A differentiated between agonistic and antagonistic ligands. On the basis of the high-resolution X-ray structure of bovine rhodopsin as well as available data on GnRH-R mutants, models for ligand-receptor interactions are proposed. The model for D-Trp(6)-GnRH (Triptorelin) binding, representing a superagonistic ligand, is in full accordance to available data. Furthermore, new interactions are proposed: pGlu(1) interacts with N(212) in transmembrane helix 5, Tyr(5) with Y(290), and D-Trp(6) with W(289). The binding behavior of mutants W(289)A and Y(290)A corresponds to the proposed binding model for the antagonist Cetrorelix. In summary, our data as presented indicate that Y(290) plays a key function in agonist but not antagonist binding.     

Reciprocal mutations of highly conserved residues in transmembrane helices 2 and 7 of the alpha(2A)-adrenoceptor restore agonist activation of G(i1)alpha.

Fusion proteins were constructed between the alpha(2A)-adrenoceptor and the alpha-subunit of the G-protein G(i1). Mutation of the highly conserved Asp(79) in transmembrane (TM) helix 2 of the receptor to Asn reduced the capacity of agonists to activate G(i1)alpha by 95% without altering [3H]antagonist or agonist ligand-binding affinity. A reciprocal mutation in TM helix 7 (Asn(422)Asp) was without effect on signalling effectiveness. Combination of these two mutations overcame the effect of the Asp(79)Asp mutation. By examining alterations in this helix 2-helix 7 microdomain, we further demonstrate the utility of receptor-G-protein fusion proteins to quantitate mutational effects on receptor-G-protein interactions and information transfer.     

Role of key aromatic residues in the ligand-binding domain of alpha7 nicotinic receptors in the agonist action of beta-amyloid

Soluble β-amyloid (Aβ) resides in certain regions of the brain at or near picomolar concentration, rising in level during the prodromic stage of Alzheimer disease. Recently, we identified the homomeric α7 nicotinic acetylcholine receptor (α7-nAChR) as one possible functional target for picomolar Aβ. This study was aimed at addressing which residues in α7-nAChRs potentially interact with Aβ to regulate the presynaptic function of this receptor. Site-directed mutagenesis was carried out to study the key aromatic residues in the mouse α7-nAChR agonist-binding pocket. Mutations of tyrosine188 resulted in a decrease in activation of presynaptic α7-nAChRs by ACh and Aβ but with no change in response to nicotine, indicating the critical role of Tyr-188 in presynaptic regulation by Aβ. Coimmunoprecipitation additionally revealed direct binding of Aβ to α7-nAChRs and to the Tyr-188 mutant receptor. In contrast, mutations of Tyr-195 in α7-nAChR led to decreased activation by nicotine without apparent effects on ACh- or Aβ-induced responses. Agonist-induced responses of Tyr-93 mutant α7-nAChRs indicated possible interactions of nicotine and Aβ with its hydroxyl group, but there was no change in presynaptic responses after mutation of Trp-149. All of the mutants were shown to be expressed on the plasma membrane using cell surface labeling. Together, these results directly demonstrate an essential role for the aromatic residue Tyr-188 as a key component in the agonist binding domain for the activation of α7-nAChRs by Aβ.     

1H NMR (500 MHz) identification of aromatic residues of gene 32 protein involved in DNA binding by use of protein containing perdeuterated aromatic residues and by site-directed mutagenesis

Preparation of gene 32 protein containing perdeuterated tyrosyl and phenylalanyl residues has allowed the resolution of separate 1H NMR signals for the Tyr and Phe residues of the protein by NMR difference spectra. Upfield shifts in the chemical shifts of a number of aromatic protons previously observed to accompany deoxyoligonucleotide complex formation with gene 32 protein [Prigodich, R. V., Casas-Finet, J., Williams, K. R., Konigsberg, W., & Coleman, J. E. (1984) Biochemistry 23, 522-529] can be assigned to five Tyr and two Phe residues that must form part of the DNA binding domain. Site-directed mutation of Tyr-115 to Ser-115 results in the disappearance of a set of 2,6 and 3,5 tyrosyl protons that are among those moved upfield by oligonucleotide complex formation. These findings suggest that the amino acid sequence from Tyr-73 to Tyr-115 which contains six of the eight Tyr residues of the protein forms part of the DNA binding surface.     

Regional distribution of rat brain alpha 1-adrenergic receptors: correlation between (125I)-heat membrane binding and in vitro autoradiography

[125I]-HEAT has proven useful for in vitro autoradiography as a specific alpha 1-adrenergic radioligand. We compared the binding of [125I]-HEAT to membranes from ten brain regions with the densitometric readings of these regions in autoradiographs. There was an excellent correlation between receptor numbers from membrane binding and relative optical densities from the autoradiography. The affinity of HEAT for binding to membranes from various regions was similar. The results of this direct comparison are further evidence that HEAT binds to alpha 1-adrenergic receptors in lightly fixed tissue sections. A further interesting observation is that in regions with a heterogeneous distribution of binding sites, membrane binding may not reflect the presence of a dense local population of receptors.     

Integrin LFA-1 alpha subunit contains an ICAM-1 binding site in domains V and VI.

In order to identify a binding site for ligand intercellular adhesion molecule-1 (ICAM-1) on the beta 2 integrin lymphocyte function-associated antigen-1 (LFA-1), protein fragments of LFA-1 were made by in vitro translation of a series of constructs which featured domain-sized deletions starting from the N-terminus of the alpha subunit of LFA-1. Monoclonal antibodies and ICAM-1 were tested for their ability to bind to these protein fragments. Results show that the putative divalent cation binding domains V and VI contain an ICAM-1 binding site. A series of consecutive peptides covering these domains indicated two discontinuous areas as specific contact sites: residues 458-467 in domain V and residues 497-516 in domain VI. A three-dimensional model of these domains of LFA-1 was constructed based on the sequence similarity to known EF hands. The two regions critical for the interaction of LFA-1 with ICAM-1 lie adjacent to each other, the first next to the non-functional EF hand in domain V and the second coinciding with the potential divalent cation binding loop in domain VI. The binding of ICAM-1 with the domain V and VI region in solution was not sensitive to divalent cation chelation. In short, a critical motif for ICAM-1 binding to the alpha subunit of LFA-1 is shared between two regions of domains V and VI.     

Thiol cross-linking of transmembrane domains IV and V in the lactose permease of Escherichia coli

Glu126 (helix IV) and Arg144 (helix V) in the lactose permease of Escherichia coli are critical for substrate binding and transport, and the two residues are in close proximity and charge-paired. By using a functional permease construct with two tandem factor Xa protease sites in the cytoplasmic loop between helices IV and V, it is shown here that Cys residues in place of Glu126 and Arg144, as well as Ala122 and Val149, spontaneously form disulfide bonds in situ, indicating that this region of transmembrane domains IV and V is in the alpha-helical conformation. To determine if the local structure or environment is perturbed by the presence of an unpaired charge, either Glu126 or Arg144 or both were replaced with Ala, and cross-linking between the Cys pair Ala122-->Cys/Val149-->Cys was studied. Ala replacement for Arg144 causes a marked decrease in cross-linking, while Ala replacement for Glu126 alone or for both Glu126 and Arg144 has little effect. The data provide strong support for the argument that Glu126 and Arg144 are within close proximity and suggest that an unpaired carboxylate at position 126 causes a structural change at the interface between helices IV and V.     

Interaction of SNX482 with domains III and IV inhibits activation gating of alpha(1E) (Ca(V)2.3) calcium channels.

We have investigated the action of SNX482, a toxin isolated from the venom of the tarantula Hysterocrates gigas, on voltage-dependent calcium channels expressed in tsa-201 cells. Upon application of 200 nM SNX482, R-type alpha(1E) calcium channels underwent rapid and complete inhibition, which was only poorly reversible upon washout. However, upon application of strong membrane depolarizations, rapid and complete recovery from inhibition was obtained. Tail current analysis revealed that SNX482 mediated an approximately 70 mV depolarizing shift in half-activation potential, suggesting that the toxin inhibits alpha(1E) calcium channels by preventing their activation. Experiments involving chimeric channels combining structural features of alpha(1E) and alpha(1C) subunits indicated that the presence of the domain III and IV of alpha(1E) is a prerequisite for a strong gating inhibition. In contrast, L-type alpha(1C) channels underwent incomplete inhibition at saturating concentrations of SNX482 that was paralleled by a small shift in half-activation potential and which could be rapidly reversed, suggesting a less pronounced effect of the toxin on L-type calcium channel gating. We conclude that SNX482 does not exhibit unequivocal specificity for R-type channels, but highly effectively antagonizes their activation.     

Evidence for cooperative binding of (-)Isoproterenol to rat brain beta1-adrenergic receptors

In the present study, the effects of the thiol oxidising agent diamide upon the properties of rat brain beta1-adrenergic and human platelet serotonin2A receptor recognition sites have been investigated using [3H](-)CGP-12177 (in the presence of ICI-118551) and [3H]LSD as ligands. (-)Isoprenaline inhibited [3H](-)CGP-12177 binding with nH values of 0.87, 0.67, and 0.56 for added Mg2+ concentrations of 0, 2.5, and 25 mM, respectively. Pretreatment with diamide increased the nH to above unity for the inhibition of the binding by (-)isoprenaline, without a concomitant effect on the inhibition of the binding by (-)propranolol. In contrast, diamide reduced the affinity of human platelet serotonin2A-receptors for antagonists, but did not consistently induce nH values above unity for the inhibition of antagonist binding by serotonin. These results suggest that cooperative interactions reported for cardiac muscarinic receptors may also occur for beta1-adrenergic receptors in the rat brain.     

Adverse effects of constitutively active alpha(1B)-adrenergic receptors after pressure overload in mouse hearts

Cardiac hypertrophy and function were studied 6 wk after constriction of the thoracic aorta (TAC) in transgenic (TG) mice expressing constitutively active mutant alpha(1B)-adrenergic receptors (ARs) in the heart. Hearts from sham-operated TG animals and nontransgenic littermates (WT) were similar in size, but hearts from TAC/TG mice were larger than those from TAC/WT mice, and atrial natriuretic peptide mRNA expression was also higher. Lung weight was markedly increased in TAC/TG animals, and the incidence of left atrial thrombus formation was significantly higher. Ventricular contractility in anesthetized animals, although it was increased in TAC/WT hearts, was unchanged in TAC/TG hearts, implying cardiac decompensation and progression to failure in TG mice. There was no increase in alpha(1A)-AR mRNA expression in TAC/WT hearts, and expression was significantly reduced in TAC/TG hearts. These findings show that cardiac expression of constitutively actively mutant alpha(1B)-ARs is detrimental in terms of hypertrophy and cardiac function after pressure overload and that increased alpha(1A)-AR mRNA expression is not a feature of the hypertrophic response in this murine model.     

Synthesis of new piperazine-pyridazinone derivatives and their binding affinity toward alpha1-, alpha2-adrenergic and 5-HT1A serotoninergic receptors

We report the design and synthesis of a new class of piperazine-pyridazinone analogues. The arylpiperazine moiety, the length of the spacer, and the terminal molecular fragment were varied to evaluate their influence in determining the affinity of the new compounds toward the alpha1-adrenergic receptor (alpha1-AR), alpha2-adrenergic receptor (alpha2-AR), and the 5-HT1A serotoninergic receptor (5-HT1AR). Biological data showed that most of the compounds have an alpha1-AR affinity in the nanomolar or subnanomolar range, while affinity toward the other two receptors was lower in most cases. However, several of the tested compounds also showed very good (in the nanomolar range) or moderate affinity toward the 5-HT1AR subtype.     

Non-charged amino acids from three different domains contribute to link agonist binding to channel gating in alpha7 nicotinic acetylcholine receptors

Binding of agonists to nicotinic acetylcholine receptors results in channel opening. Previously, we have shown that several charged residues at three different domains of the alpha7 nicotinic receptor are involved in coupling binding and gating, probably through a network of electrostatic interactions. This network, however, could also be integrated by other residues. To test this hypothesis, non-charged amino acids were mutated and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Asn47 and Gln48 (loop 2), Ile130, Trp134, and Gln140 (loop 7), and Thr264 (M2-M3 linker) showed poor or null functional responses, despite significant membrane expression. By contrast, mutants F137A and S265A exhibited a gain of function effect. In all cases, changes in dose-response relationships were small, EC(50) values being between threefold smaller and fivefold larger, arguing against large modifications of agonist binding. Peak currents decayed at the same rate in all receptors except two, excluding large effects on desensitization. Thus, the observed changes could be mostly caused by alterations of the gating characteristics. Moreover, analysis of double mutants showed an interconnection between some residues in these domains, especially Gln48 with Ile130, suggesting a potential coupling between agonist binding and channel gating through these amino acids.