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The α1a and α1b-adrenergic receptor subtypes: molecular mechanisms of receptor activation and of drug action
Pharmaceutica Acta Helvetiae 74 Ž2000. 173–179 www.elsevier.comrlocaterpharmactahelv
The a 1a and a 1b-adrenergic receptor subtypes: molecular mechanisms of receptor activation and of drug action Susanna Cotecchia a
a,)
, Olivier Rossier a , Francesca Fanelli b, Amedeo Leonardi c , Pier G. De Benedetti b
Institut de Pharmacologie et Toxicologie, UniÕersite´ de Lausanne, 27, Rue du Bugnon, Faculte´ de Medecine, 1005 Lausanne, Switzerland ´ b Dipartimento di Chimica, UniÕersita` di Modena e Reggio Emilia, 41100 Modena, Italy c Pharmaceutical R&D DiÕision, Recordati, 20148 Milan, Italy
Abstract In this chapter we summarize some aspects of the structure-functional relationship of the a1a and a1b-adrenergic receptor subtypes related to the receptor activation process as well as the effect of different alpha-blockers on the constitutive activity of the receptor. Molecular modeling of the a1a and a1b-adrenergic receptor subtypes and computational simulation of receptor dynamics were useful to interpret the experimental findings derived from site directed mutagenesis studies. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Adrenergic receptors; G protein-coupled receptors; Constitutive activity; Inverse agonism; Molecular dynamics
1. The a 1-adrenergic receptor subtypes The receptors for a large number of hormones and neurotransmitters regulate cellular activity via the intermediary role of guanine nucleotide regulatory proteins ŽG proteins.. Among these receptors, the adrenergic receptors ŽAR. mediate the effects of epinephrine and norepinephrine by coupling to several of the major signalling pathways modulated by G protein. The AR family includes nine different gene products: three b Žb 1 , b 2 , b 3 ., three a 2 Ž a 2A , a 2B , a 2C . and three a 1 Ž a 1a , a 1b , a 1d . receptor subtypes. The a 1-AR is present in many tissues including brain, heart, blood vessels, liver, kidney, prostate and spleen. In these tissues the a 1-ARs mediate a variety of physiological effects such as neurotransmission, vasoconstriction, cardiac inotropy and chronotropy, glycogenolysis Žfor reviews, see Michel et al., 1995; Graham et al., 1996.. Radioligand binding studies in different rat tissues have demonstrated two classes of a 1-AR binding sites, ‘‘A’’ and ‘‘B’’ with high and low affinity for the a-AR antagonists WB4101 and phentolamine, respectively. After large scale purification of the a 1-AR from smooth muscle cells DDT1 MF-2 a first receptor was cloned, unequivocally )
Corresponding author. Tel.: q41-21-692-5400; fax: q41-21-6925355; e-mail: [email protected]
assigned to the pharmacological a 1B subtype and hence named a 1b -AR. On the other hand, the pharmacological a 1A subtype is mainly coded by the a 1a-AR initially cloned from a bovine brain library and inappropriately named a 1C -AR. Finally, the cloned a 1d-AR Žinitially named a 1A -AR or a 1ArD -AR. represents a receptor subtype not clearly identified by previous pharmacological studies. Activation of the a 1-ARs causes polyphosphoinositide hydrolysis catalyzed by phospholipase C via pertussis toxin-insensitive G proteins in almost all tissues where this effect has been examined. Recent studies have shown that other signalling pathways can be activated upon a 1-AR stimulation such as phosphatidylcholine hydrolysis and phospholipase A2 Žfor review, see Graham et al., 1996.. However, the comparison among different a 1-AR-mediated responses in various tissues has not allowed to assess any conclusive signalling differences among distinct a 1AR subtypes. In vivo studies aiming to assess a specificity of the functional responses mediated by distinct a 1-AR subtypes have been hampered by the fact that the subtype-selective drugs are only moderately selective and might interact with both adrenergic and non-adrenergic receptors. To contribute to the elucidation of the physiological role of the a 1-AR subtypes in vivo we have used gene targeting to create knockout mice lacking the a 1b -AR ŽCavalli et al.,
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1997.. Our findings provide strong evidence that the a 1b AR can be a mediator of the blood pressure response as well as of the aorta contractility induced by a 1-agonists. A full understanding of the functional implications of adrenergic receptor heterogeneity awaits the knock out of all AR subtypes as well as the intercross among different knock out models.
2. Structure-functional analysis of the a 1-AR subtypes Like all G protein-coupled receptors ŽGPCRs., the a 1AR subtypes share the presence of seven hydrophobic regions which form transmembrane ŽTM. a-helices and are connected by alternating intracellular Ži. and extracellu-
lar Že. hydrophilic loops ŽFig. 1.. Mutational analysis of several GPCRs has revealed that the a-helical bundle contributes to form the ligand binding site of the receptor, whereas amino acid sequences of the intracellular regions appear to mediate the interaction of the receptor with G proteins as well as with different signalling and regulatory proteins ŽWess, 1997.. Recent studies have focused on the molecular interactions of the endogenous catecholamines, epinephrine and norepinephrine, with the a 1a and a 1b-AR subtypes ŽCavalli et al., 1996; Hwa and Perez, 1996.. Epinephrine and norepinephrine contain a protonated nitrogen atom separated from the aromatic catechol ring by a b-hydroxylethyl chain. The molecular requirement for catecholamine binding to the AR should include the electrostatic interaction between the receptor and the amino group of the ligand,
Fig. 1. Average minimized structures of the wild type a 1 -AR subtypes and of their constitutively active mutants. Views of the receptor structures are in a direction parallel to the membrane surface with the extracellular side at the bottom. The figure shows also the interactions involving some of the conserved polar amino acids including the arginine of the ErDRY motif ŽR124 in the a 1a -AR and R143 in the a 1b-AR..
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hydrogen bonds between donorracceptor sites of the receptor and the b-hydroxyl as well as the catechol metaand para-hydroxyl groups of the ligand, and finally van der Waals attractive interactions. Mutagenesis studies of the a 1b -AR subtypes suggested that the amino group of the catecholamines as well as of the a 1-antagonists makes an electrostatic interaction with the carboxylate side chain of an aspartate D125 on helix 3 which is highly conserved in all GPCR that bind amine ligands. On the other hand, there is evidence that the catechol meta- and para-hydroxyl groups of catecholamines interact with serine residues present in helix 5 which are also conserved in different catecholamine receptors. Our work on the a 1b -AR ŽCavalli et al., 1996. suggested that S207 in helix 5 interacts with both catecholic hydroxy-groups of Žy.-epinephrine. In contrast, in the a 1a -AR of the two serines S188 and S192 present on helix 5, S188 seems to form one, but not two hydrogen bondings with the catechol ring ŽHwa and Perez, 1996.. Very little is known so far about the receptor amino acids which interact with different antagonists as well as about the structural basis underlying receptor selectivity for different ligands and pharmacological agents. Some information has been gained about the domains of the a 1b -AR involved in receptor coupling to the G protein. Studies with chimeric b 2ra 1b-AR receptors indicated that a stretch of 27 amino acids Žresidues 233–259. derived from the N-terminal portion of the third intracellular loop of the a 1b -AR were sufficient to confer to the b 2-AR the ability to activate phospholipase C ŽCotecchia et al., 1992.. Virtually nothing is known about the structural determinants of the other a 1-AR subtypes involved in Gq-mediated activation of phospholipase C.
3. Molecular modelling of the a 1-AR subtypes To probe the structure-function relationships of GPCRs several useful molecular models of these membrane proteins were built using different methods. To investigate the potential intramolecular motions underlying different functional states of the a 1-AR subtypes, we combined 3-D model building of the receptor structure with computational simulation of receptor dynamics as recently described ŽFanelli et al., 1998.. The a 1-AR model was built using an iterative ab initio procedure started with a comparative molecular dynamics ŽMD. study on the helix bundle of seven GPCRs Ž a 1b -AR, b 2-AR, a 2-AR, dopamine D 2 , 5-HT1A , muscarinic M 1 and the bovine rhodopsin not complexed with retinal.. The arrangement of the helices was based on the structural constraints inferred from the analysis of a large number of GPCR sequences ŽBaldwin et al., 1997.. The a 1b -AR model was progressively upgraded on the basis of the new structural information derived from the electron micrographs of 3-D frog rhodopsin crystals ŽUnger et al., 1997.. In addition, the model was completed by adding the intracellular and
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extracellular loops as shown in Fig. 1 ŽScheer et al., 1996; Fanelli et al., 1998.. Packing of the seven helices of the a 1a -AR was achieved by using the last upgraded input structure of the a 1b -AR as a template. The seven helix bundle of the a 1a -AR in the input arrangement was then completed with the extracellular and intracellular loops according to the procedure recently described ŽFanelli et al., 1998, 1999.. The analysis of the MD trajectories was used to compare the structuralrdynamic features of functionally different receptor mutants with those of the wild type a 1a and a 1b-AR, to predict key residues the mutations of which would either activate or inactivate the receptor and to investigate the potential effects of mutations.
4. The activation process of the a 1a and a 1b-AR subtypes Agonist binding to a GPCR is believed to induce a conformational change of the receptor which results in its productive coupling to heterotrimeric G proteins thus leading to intracellular signalling events. Yet, very little is known on how binding of the agonist triggers receptor activation, i.e., the ‘‘conformational switch’’ of the receptor leading to productive receptor-G protein coupling. An important contribution to our understanding of receptor activation has been provided by the findings that point mutations in a GPCR could increase the constitutive Žagonist-independent. activity of the receptor. In the a 1b AR, all possible amino acid substitutions of A293 in the C-terminal portion of the i3 induced variable levels of constitutive activity ŽKjelsberg et al., 1992.. Similar mutations were also found to increase the constitutive activity of the b 2-AR and a 2-AR ŽRen et al., 1993; Samama et al., 1993a,b.. A detailed analysis of the properties of the constitutively active AR mutants was instrumental to propose the ‘‘allosteric ternary complex’’ model to describe GPCR behaviour ŽSamama et al., 1993a,b.. This model predicts that GPCRs can undergo an allosteric transition between at least two interconvertible states, R Žinactive or ground state. and RU Žactive state.. In the absence of the agonist, R predominates possibly because a structural constraint might prevent sequences of the intracellular loops to interact with the G proteins. On the other hand, agonists can trigger the equilibrium toward RU thus favouring its stabilization. Constitutively activating mutations might trigger the formation of RU releasing the structural constraint which keeps GPCR inactive. These findings led to the hypothesis that activating mutations mimic, at least to some extent, the conformational change triggered by agonist binding to GPCR. Thus, the identification of receptor sites susceptible to constitutive activation might contribute to elucidate the structural basis of the ‘‘conformational switch’’ underlying GPCR activation.
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Two residues seem to play a crucial role in the activation process of the a 1b -AR subtype, D142 belonging to the highly conserved ErDRY motif lying at the end of helix 3 and A293 in the C-terminal portion of the 3i loop ŽFig. 1.. This is mainly supported by the findings that mutations of D142 as well as of A293 could markedly increase the constitutive activity of the a 1b -AR ŽKjelsberg et al., 1992; Scheer et al., 1997.. Recently, we have shown that in a 1a -AR mutations of D123 into alanine and of A271 into glutamic acid Žwhich are homologous to D142 and A293 of the a 1b -AR, respectively. could also increase the constitutive activity of the receptor ŽFig. 2. ŽRossier et al., 1999.. Altogether these findings suggest that the ErDRY motif as well as the C-terminal end of the 3i loop plays a role in the ‘‘conformational switch’’ underlying the activation process of both the a 1a and a 1b-AR subtypes. The role played by these sequences in receptor activation is probably shared by other members of the rhodopsin family of GPCRs as supported by the fact that mutations in the homologous regions of other receptors can have marked effects on receptor-G protein coupling ŽWess, 1997.. The comparative MD analysis of the wild type a 1-AR subtypes and of a number of functionally different receptor mutants was instrumental to make hypothesis about the potential molecular mechanisms underlying the transition between the inactive ŽR. and active states ŽRU . of the receptor. Our findings highlighted a network of hydrogenbonding interactions among some conserved polar residues forming a ‘‘polar pocket’’ near the cytosol ŽFig. 1. and the arginine of the ErDRY motif. We suggested that this set of interactions constrains the receptor in its ground or inactive state ŽR. by controlling the degree of cytosolic exposure attainable by the arginine of the ErDRY sequence. This hypothesis is supported by the observation that the charge reinforced H-bonding interaction between the conserved aspartate on helix 2 and the arginine of the ErDRY sequence constitutes one of the constraining interactions in the receptor ground state. In contrast, a landmark of the constitutively active receptor mutants is the
breakage of this interaction and the shift of the arginine out of the ‘‘polar pocket’’ ŽFig. 1.. The results of the MD simulations indicate that the motions of the helix bundle triggered by different activating mutations in the a 1-AR subtypes induce the breakage of the interactions within the ‘‘polar pocket’’ and a rearrangement of the cytosolic domains which seem to form a site with docking complementarity with the G protein ŽFig. 1. ŽFanelli et al., 1999.. Our findings suggest that the main role of the arginine of the ErDRY motif is to mediate receptor activation either directly interacting with the G protein heterotrimer or allowing several cationic amino acids in i2 and i3 to attain the right configuration for the formation of a site with docking complementarity with the G protein. This hypothesis is supported by the experimental findings showing that different mutations of R143 can almost entirely inactivate the a 1b -AR ŽScheer et al., 1996.. 5. Inverse agonism and neutral antagonism at the a 1a and a 1b -AR subtypes Both selective and nonselective antagonists for different AR subtypes are widely used in a variety of pathological conditions including hypertension, heart failure, prostate hypertrophy as well as in mental diseases such as depression. Several studies have demonstrated that beta-blockers can behave either as neutral antagonists or inverse agonists at the wild type b 2-AR or at a constitutively active b 2-AR mutant ŽSamama et al., 1993a,b; Chidiac et al., 1994.. On the other hand, inverse agonism at other AR subtypes has been less extensively investigated. It has been previously reported that a small range of alpha-blockers could inhibit the agonist-independent phospholipase C as well as phospholipase D responses mediated by constitutively active mutants of the a 1b -AR ŽLee et al., 1997.. A recent study has demonstrated that some alpha-blockers can inhibit the spontaneous activity of the a 1d -AR subtype ŽGarcıa-Sainz ´ ´ and Torres-Padilla, 1999..
Fig. 2. Constitutively active a 1a -AR and a 1b-AR mutants. D123 and A271 of the a 1a-AR as well as D142 and A293 of the a 1b-AR have been mutated into alanine and glutamic acid, respectively. Receptors expression in COS-7 cells ranged 1.5–2.5 pmolrmg of protein. Inositol phosphate Žw3 HxIP. accumulation was measured in cells expressing the wild type or mutated receptors after incubation in the absence ŽBasal. or presence of 10y4 M epinephrine for 45 min. Results are the mean"S.E of three independent experiments.
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We have recently characterized the pharmacological antagonism, i.e., neutral antagonism or inverse agonism, displayed by a number of alpha-blockers at two a 1-AR subtypes, the a 1a and a 1b-AR ŽRossier et al., 1999.. One strategy to identify inverse agonists is to screen drugs for their ability to inhibit the agonist-independent activity of the constitutively active receptor mutants. Thus, 24 alphablockers differing in their chemical structures were tested for their effect on the basal activity of the constitutively active A271E and A293E mutants expressed in COS-7 cells ŽFig. 3.. All the ligands used in this study, except REC 15r3039, were previously described for their structure, their binding affinity at recombinant as well as native a 1-AR subtypes and some of their pharmacological effects in different tissues ŽMichel et al., 1995; Leonardi et al., 1997; Testa et al., 1997.. Our results show that the vast majority of alpha-blockers displayed inverse agonism as demonstrated by their
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ability to decrease the basal activity of both CAMs. However, the various alpha-blockers differed in their negative efficacy and some of these differences depended on the a 1-AR subtype. Drugs with the highest negative efficacy Ždefined as G 70% inhibition of the basal activity. at both CAMs included WAY 100635, WB 4101, all the tested quinazolines Žprazosin, terazosin, both Žq.- and Žy.cyclazosin, REC 15r2615 and alfuzosin., indoramin, corynanthine, spiperone and AH11110A ŽFig. 3.. For the other drugs, their negative efficacy differed at the two CAMs. The most striking difference concerned some N-arylpiperazines which displayed modest negative efficacy Že.g., 5-methylurapidil, BMY 7378 and REC 15r2869. or neutral antagonism Že.g., REC 15r3039, REC 15r2739 and REC 15r3011. at the A271E mutant. On the other hand, the negative efficacy of these compounds was more pronounced at the A293E resulting in at least 45% inhibition of the receptor-mediated basal activity. For phentolamine,
Fig. 3. Ligand-induced inhibition of the basal inositol phosphate response mediated by constitutively active a 1 -AR mutants. Inositol phosphate Žw3 HxIP. accumulation was measured in COS-7 cells expressing the wild type ŽWT. a 1a -AR and a 1b -AR and their respective mutants A271E and A293E. In cells expressing the receptor mutants, w3 HxIP were measured in the absence ŽBasal. or presence of different ligands at a concentration of 10y5 M Ž10y4 M for REC 15r3039. for 45 min. Mock indicates cells not expressing the receptors. The ligands are grouped according to their structural similarities Žfrom left to right the groups include: N-arylpiperazines; 1,4-dihydropyridines; imidazolines; benzodioxanes and phenylalkylamines; quinazolines; various structures.. Results are the mean"S.E of three to six independent experiments.
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BE 2254 and tamsulosin negative efficacy was also greater at the A293E than at the A271E. In contrast, for Ž S .-Žq.niguldipine negative efficacy was greater at the A293E than at the A271E mutant. To assess whether the effect of the alpha-blockers observed on the CAMs reflected their behaviour at the wild type receptors, a selected number of drugs were tested for their effects in COS-7 cells expressing the wild type a 1a and a 1b -AR subtypes. Overexpression of both a 1-AR subtypes resulted in a small, but measurable increase of agonist-independent accumulation of IP which was greater for the a 1b -AR than for the a 1a-AR. The effect of the alpha-blockers in cells expressing the wild type a 1-AR subtypes displayed several similarities to that observed in cells expressing their CAMs. Altogether, our findings identify a group of N-arylpiperazines as alpha-blockers which display the most striking difference at the two a 1-AR subtypes being inverse agonists with profound negative efficacy at the wild type a 1b -AR, but not at the a 1a-AR. In particular, REC 15r3039, REC 15r2739 and REC 15r3011 are the first alpha-blockers identified so far which do not display inverse agonism at one of the a 1-AR subtypes, namely, the a 1a . A preliminary structure-activity relationship analysis of the ligands suggests that the inhibitory effect observed for different ligands on the constitutive activity of the a 1a -AR and, to a smaller extent, on that of the a 1b -AR is related to the structure of a defined portion of the ligands. Our results suggest that the geometry of the protonated nitrogen atom as well as that of the molecular moiety closest to this nitrogen might be responsible for the functional effect of the ligands tested ŽRossier et al., 1999.. The group of quinazolines including prazosin, terazosin and alfuzosin, which are among the most commonly used alpha-blockers, can display unwanted effects in vivo on the cardiovascular system such as orthostatic hypotension. In contrast, REC 15r2739, REC 15r2869 and REC 15r3011 are characterized by high selectivity for the urogenital tissues and seem to have less pronounced effects on the cardiovascular system in vivo ŽTesta et al., 1997.. In future studies, it will be interesting to identify novel alpha-blockers which are neutral antagonists at one or more a 1-AR subtypes and to assess whether these compounds, including REC 15r3039, have fewer generalized cardiovascular effects as compared to full inverse agonists.
6. Conclusions. The results of our recent studies provide new information on the activation process of the a 1a and a 1b-AR subtypes as well as on the mechanisms of action of drugs acting at these receptors. They might also provide generalities about the molecular mechanisms underlying the activation process of other GPCRs. The structural conservation
among different receptors suggests that the molecular mechanisms proposed for the a 1a - and a 1b-AR subtypes might be generalized to the activation process of other members of the GPCR family. We propose that an interdisciplinary approach combining site-directed mutagenesis, molecular dynamics and thermodynamical analysis of receptor mutants is useful to elucidate the structural-dynamic properties of GPCRs.
Acknowledgements This work was supported by the Fonds National Suisse de la Recherche Scientifique Žgrant 31-51043.97. and by the European Community Žgrant BMH4-CT97-2152..
References Baldwin, J.M., Schertler, G.F., Unger, V.M., 1997. An a-carbon template for the transmembrane helices in the rhodopsin family of G proteincoupled receptors. J. Mol. Biol. 272, 144–164. Cavalli, A., Fanelli, F., Taddei, C., De Benedetti, P.G., Cotecchia, S., 1996. Amino acids involved in agonist binding: differences in docking catecholamines to receptor subtypes. FEBS Lett. 399, 9–13. Cavalli, A., Lattion, A.L., Hummler, E., Nenniger, M., Pedrazzini, T., Aubert, J.-F., Michel, M., Ming, Y., Lembo, G., Vecchione, C., Mostardini, M., Schmidt, A., Beermann, F., Cotecchia, S., 1997. Decreased blood pressure response in mice deficient of the a 1B adrenergic receptor. Proc. Natl. Acad. of Sci. USA 94, 11589. Chidiac, P., Hebert, T.E., Valiquette, M., Dennis, M., Bouvier, M., 1994. Inverse agonist activity of b-adrenergic antagonists. Mol. Pharmacol. 45, 490–499. Cotecchia, S., Ostrowski, J., Kjelsberg, M.A., Caron, M.G., Lefkowitz, R.J., 1992. Discrete amino acid sequences of the a 1 -adrenergic receptor determine the selectivity of coupling to phosphatidylinositol hydrolysis. J. Biol. Chem. 267, 1633–1639. Fanelli, F., Menziani, M.C., Scheer, A., Cotecchia, S., De Benedetti, P.G., 1998. Ab initio modeling and molecular dynamics simulation of the a 1b -adrenergic receptor activation. Methods Companion Methods Enzymol. 14, 302–317. Fanelli, F., Menziani, C., Scheer, A., Cotecchia, S., De Benedetti, P.G., 1999. Theoretical study on receptorrG protein recognition: new insights into the mechanism of the a 1b -adrenergic receptor activation. Int. J. Quantum Chem. 73, 71–83. Garcıa-Sainz, J.A., Torres-Padilla, M.E., 1999. Modulation of basal ´ ´ intracellular calcium by inverse agonists and phorbol myristate acetate in rat-1 fibroblasts stably expressing a 1d -adrenoceptors. FEBS Lett. 443, 277–281. Graham, R.M., Perez, D.M., Hwa, J., Piascik, M.T., 1996. a 1-Adrenergic receptor subtypes, molecular structure, function and signalling. Circ. Res. 78, 737–749. Haw, J., Perez, D.M., 1996. The unique nature of the serine interactions for a 1-adrenergic receptor agonist binding and activation. J. Biol. Chem. 271, 6322–6327. Kjelsberg, M.A., Cotecchia, S., Ostrowski, J., Caron, M.G., Lefkowitz, R.J., 1992. Constitutive activation of the alpha 1B-adrenergic receptor by all amino acid substitutions at a single site. Evidence for a region which constrains receptor activation. J. Biol. Chem. 267, 1430–1433. Lee, T.W., Cotecchia, S., Milligan, G., 1997. Up-regulation of the levels of expression and function of a constitutively active mutant of the hamster a 1B -adrenoceptor by ligands that act as inverse agonists. Biochem. J. 325, 733–739.
S. Cotecchia et al.r Pharmaceutica Acta HelÕetiae 74 (2000) 173–179 Leonardi, A., Hieble, J.P., Guarneri, L., Naselsky, D.P., Poggesi, E., Sironi, G., Sulpizio, A.C., Testa, R., 1997. Pharmacological characterization of the uroselective alpha-1 antagonist Rec 15r2739 ŽSB 216469.: role of the alpha-1L adrenoceptor in tissue selectivity, part I. J. Pharmacol. Exp. Ther. 281, 1272–1283. Michel, M.C., Kenny, B., Schwinn, D.A., 1995. Classification of a 1adrenoceptor subtypes. Naunyn-Schmiedeberg’s Arch. Pharmacol. 352, 1–10. Ren, Q., Kurose, H., Lefkowitz, R.J., Cotecchia, S., 1993. Constitutively active mutants of the a 2 -adrenergic receptor . J. Biol. Chem. 268, 16483–16487. Rossier, O., Abuin, L., Fanelli, F., Leonardi, A., Cotecchia, S., 1999. Inverse agonism and neutral antagonism at the a 1a and a 1b adrenergic receptor subtypes. Mol. Pharmacol. Žin press.. Samama, P., Cotecchia, S., Costa, T., Lefkowitz, R.J., 1993a. A mutation-induced activated state of the b 2 -adrenergic receptor. Extending the ternary complex model. J. Biol. Chem. 268, 4625–4636.
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Samama, P., Pei, G., Costa, T., Cotecchia, S., Lefkowitz, R.J., 1993b. Negative antagonists promote an inactive conformation of the b 2 adrenergic receptor. Mol. Pharmacol. 45, 390–394. Testa, R., Guarneri, L., Angelico, P., Poggesi, E., Taddei, C., Sironi, G., Colombo, D., Sulpizio, A.C., Naselsky, D.P., Hieble, J.P., Leonardi, A., 1997. Pharmacological characterization of the uroselective alpha-1 antagonist Rec 15r2739 ŽSB 216469.: role of the alpha-1L adrenoceptor in tissue selectivity, part II. J. Pharmacol. Exp. Ther. 281, 1284–1293. Unger, V.M., Hargrave, P.A., Baldwin, J.M., Schertler, G.F., 1997. Arrangement of rhodopsin transmembrane alpha-helices. Nature 389, 203–206. Wess, J., 1997. G-protein-coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G-protein recognition. FASEB J. 5, 346–354.
The a 1a and a 1b-adrenergic receptor subtypes: molecular mechanisms of receptor activation and of drug action Susanna Cotecchia a
a,)
, Olivier Rossier a , Francesca Fanelli b, Amedeo Leonardi c , Pier G. De Benedetti b
Institut de Pharmacologie et Toxicologie, UniÕersite´ de Lausanne, 27, Rue du Bugnon, Faculte´ de Medecine, 1005 Lausanne, Switzerland ´ b Dipartimento di Chimica, UniÕersita` di Modena e Reggio Emilia, 41100 Modena, Italy c Pharmaceutical R&D DiÕision, Recordati, 20148 Milan, Italy
Abstract In this chapter we summarize some aspects of the structure-functional relationship of the a1a and a1b-adrenergic receptor subtypes related to the receptor activation process as well as the effect of different alpha-blockers on the constitutive activity of the receptor. Molecular modeling of the a1a and a1b-adrenergic receptor subtypes and computational simulation of receptor dynamics were useful to interpret the experimental findings derived from site directed mutagenesis studies. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Adrenergic receptors; G protein-coupled receptors; Constitutive activity; Inverse agonism; Molecular dynamics
1. The a 1-adrenergic receptor subtypes The receptors for a large number of hormones and neurotransmitters regulate cellular activity via the intermediary role of guanine nucleotide regulatory proteins ŽG proteins.. Among these receptors, the adrenergic receptors ŽAR. mediate the effects of epinephrine and norepinephrine by coupling to several of the major signalling pathways modulated by G protein. The AR family includes nine different gene products: three b Žb 1 , b 2 , b 3 ., three a 2 Ž a 2A , a 2B , a 2C . and three a 1 Ž a 1a , a 1b , a 1d . receptor subtypes. The a 1-AR is present in many tissues including brain, heart, blood vessels, liver, kidney, prostate and spleen. In these tissues the a 1-ARs mediate a variety of physiological effects such as neurotransmission, vasoconstriction, cardiac inotropy and chronotropy, glycogenolysis Žfor reviews, see Michel et al., 1995; Graham et al., 1996.. Radioligand binding studies in different rat tissues have demonstrated two classes of a 1-AR binding sites, ‘‘A’’ and ‘‘B’’ with high and low affinity for the a-AR antagonists WB4101 and phentolamine, respectively. After large scale purification of the a 1-AR from smooth muscle cells DDT1 MF-2 a first receptor was cloned, unequivocally )
Corresponding author. Tel.: q41-21-692-5400; fax: q41-21-6925355; e-mail: [email protected]
assigned to the pharmacological a 1B subtype and hence named a 1b -AR. On the other hand, the pharmacological a 1A subtype is mainly coded by the a 1a-AR initially cloned from a bovine brain library and inappropriately named a 1C -AR. Finally, the cloned a 1d-AR Žinitially named a 1A -AR or a 1ArD -AR. represents a receptor subtype not clearly identified by previous pharmacological studies. Activation of the a 1-ARs causes polyphosphoinositide hydrolysis catalyzed by phospholipase C via pertussis toxin-insensitive G proteins in almost all tissues where this effect has been examined. Recent studies have shown that other signalling pathways can be activated upon a 1-AR stimulation such as phosphatidylcholine hydrolysis and phospholipase A2 Žfor review, see Graham et al., 1996.. However, the comparison among different a 1-AR-mediated responses in various tissues has not allowed to assess any conclusive signalling differences among distinct a 1AR subtypes. In vivo studies aiming to assess a specificity of the functional responses mediated by distinct a 1-AR subtypes have been hampered by the fact that the subtype-selective drugs are only moderately selective and might interact with both adrenergic and non-adrenergic receptors. To contribute to the elucidation of the physiological role of the a 1-AR subtypes in vivo we have used gene targeting to create knockout mice lacking the a 1b -AR ŽCavalli et al.,
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1997.. Our findings provide strong evidence that the a 1b AR can be a mediator of the blood pressure response as well as of the aorta contractility induced by a 1-agonists. A full understanding of the functional implications of adrenergic receptor heterogeneity awaits the knock out of all AR subtypes as well as the intercross among different knock out models.
2. Structure-functional analysis of the a 1-AR subtypes Like all G protein-coupled receptors ŽGPCRs., the a 1AR subtypes share the presence of seven hydrophobic regions which form transmembrane ŽTM. a-helices and are connected by alternating intracellular Ži. and extracellu-
lar Že. hydrophilic loops ŽFig. 1.. Mutational analysis of several GPCRs has revealed that the a-helical bundle contributes to form the ligand binding site of the receptor, whereas amino acid sequences of the intracellular regions appear to mediate the interaction of the receptor with G proteins as well as with different signalling and regulatory proteins ŽWess, 1997.. Recent studies have focused on the molecular interactions of the endogenous catecholamines, epinephrine and norepinephrine, with the a 1a and a 1b-AR subtypes ŽCavalli et al., 1996; Hwa and Perez, 1996.. Epinephrine and norepinephrine contain a protonated nitrogen atom separated from the aromatic catechol ring by a b-hydroxylethyl chain. The molecular requirement for catecholamine binding to the AR should include the electrostatic interaction between the receptor and the amino group of the ligand,
Fig. 1. Average minimized structures of the wild type a 1 -AR subtypes and of their constitutively active mutants. Views of the receptor structures are in a direction parallel to the membrane surface with the extracellular side at the bottom. The figure shows also the interactions involving some of the conserved polar amino acids including the arginine of the ErDRY motif ŽR124 in the a 1a -AR and R143 in the a 1b-AR..
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hydrogen bonds between donorracceptor sites of the receptor and the b-hydroxyl as well as the catechol metaand para-hydroxyl groups of the ligand, and finally van der Waals attractive interactions. Mutagenesis studies of the a 1b -AR subtypes suggested that the amino group of the catecholamines as well as of the a 1-antagonists makes an electrostatic interaction with the carboxylate side chain of an aspartate D125 on helix 3 which is highly conserved in all GPCR that bind amine ligands. On the other hand, there is evidence that the catechol meta- and para-hydroxyl groups of catecholamines interact with serine residues present in helix 5 which are also conserved in different catecholamine receptors. Our work on the a 1b -AR ŽCavalli et al., 1996. suggested that S207 in helix 5 interacts with both catecholic hydroxy-groups of Žy.-epinephrine. In contrast, in the a 1a -AR of the two serines S188 and S192 present on helix 5, S188 seems to form one, but not two hydrogen bondings with the catechol ring ŽHwa and Perez, 1996.. Very little is known so far about the receptor amino acids which interact with different antagonists as well as about the structural basis underlying receptor selectivity for different ligands and pharmacological agents. Some information has been gained about the domains of the a 1b -AR involved in receptor coupling to the G protein. Studies with chimeric b 2ra 1b-AR receptors indicated that a stretch of 27 amino acids Žresidues 233–259. derived from the N-terminal portion of the third intracellular loop of the a 1b -AR were sufficient to confer to the b 2-AR the ability to activate phospholipase C ŽCotecchia et al., 1992.. Virtually nothing is known about the structural determinants of the other a 1-AR subtypes involved in Gq-mediated activation of phospholipase C.
3. Molecular modelling of the a 1-AR subtypes To probe the structure-function relationships of GPCRs several useful molecular models of these membrane proteins were built using different methods. To investigate the potential intramolecular motions underlying different functional states of the a 1-AR subtypes, we combined 3-D model building of the receptor structure with computational simulation of receptor dynamics as recently described ŽFanelli et al., 1998.. The a 1-AR model was built using an iterative ab initio procedure started with a comparative molecular dynamics ŽMD. study on the helix bundle of seven GPCRs Ž a 1b -AR, b 2-AR, a 2-AR, dopamine D 2 , 5-HT1A , muscarinic M 1 and the bovine rhodopsin not complexed with retinal.. The arrangement of the helices was based on the structural constraints inferred from the analysis of a large number of GPCR sequences ŽBaldwin et al., 1997.. The a 1b -AR model was progressively upgraded on the basis of the new structural information derived from the electron micrographs of 3-D frog rhodopsin crystals ŽUnger et al., 1997.. In addition, the model was completed by adding the intracellular and
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extracellular loops as shown in Fig. 1 ŽScheer et al., 1996; Fanelli et al., 1998.. Packing of the seven helices of the a 1a -AR was achieved by using the last upgraded input structure of the a 1b -AR as a template. The seven helix bundle of the a 1a -AR in the input arrangement was then completed with the extracellular and intracellular loops according to the procedure recently described ŽFanelli et al., 1998, 1999.. The analysis of the MD trajectories was used to compare the structuralrdynamic features of functionally different receptor mutants with those of the wild type a 1a and a 1b-AR, to predict key residues the mutations of which would either activate or inactivate the receptor and to investigate the potential effects of mutations.
4. The activation process of the a 1a and a 1b-AR subtypes Agonist binding to a GPCR is believed to induce a conformational change of the receptor which results in its productive coupling to heterotrimeric G proteins thus leading to intracellular signalling events. Yet, very little is known on how binding of the agonist triggers receptor activation, i.e., the ‘‘conformational switch’’ of the receptor leading to productive receptor-G protein coupling. An important contribution to our understanding of receptor activation has been provided by the findings that point mutations in a GPCR could increase the constitutive Žagonist-independent. activity of the receptor. In the a 1b AR, all possible amino acid substitutions of A293 in the C-terminal portion of the i3 induced variable levels of constitutive activity ŽKjelsberg et al., 1992.. Similar mutations were also found to increase the constitutive activity of the b 2-AR and a 2-AR ŽRen et al., 1993; Samama et al., 1993a,b.. A detailed analysis of the properties of the constitutively active AR mutants was instrumental to propose the ‘‘allosteric ternary complex’’ model to describe GPCR behaviour ŽSamama et al., 1993a,b.. This model predicts that GPCRs can undergo an allosteric transition between at least two interconvertible states, R Žinactive or ground state. and RU Žactive state.. In the absence of the agonist, R predominates possibly because a structural constraint might prevent sequences of the intracellular loops to interact with the G proteins. On the other hand, agonists can trigger the equilibrium toward RU thus favouring its stabilization. Constitutively activating mutations might trigger the formation of RU releasing the structural constraint which keeps GPCR inactive. These findings led to the hypothesis that activating mutations mimic, at least to some extent, the conformational change triggered by agonist binding to GPCR. Thus, the identification of receptor sites susceptible to constitutive activation might contribute to elucidate the structural basis of the ‘‘conformational switch’’ underlying GPCR activation.
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Two residues seem to play a crucial role in the activation process of the a 1b -AR subtype, D142 belonging to the highly conserved ErDRY motif lying at the end of helix 3 and A293 in the C-terminal portion of the 3i loop ŽFig. 1.. This is mainly supported by the findings that mutations of D142 as well as of A293 could markedly increase the constitutive activity of the a 1b -AR ŽKjelsberg et al., 1992; Scheer et al., 1997.. Recently, we have shown that in a 1a -AR mutations of D123 into alanine and of A271 into glutamic acid Žwhich are homologous to D142 and A293 of the a 1b -AR, respectively. could also increase the constitutive activity of the receptor ŽFig. 2. ŽRossier et al., 1999.. Altogether these findings suggest that the ErDRY motif as well as the C-terminal end of the 3i loop plays a role in the ‘‘conformational switch’’ underlying the activation process of both the a 1a and a 1b-AR subtypes. The role played by these sequences in receptor activation is probably shared by other members of the rhodopsin family of GPCRs as supported by the fact that mutations in the homologous regions of other receptors can have marked effects on receptor-G protein coupling ŽWess, 1997.. The comparative MD analysis of the wild type a 1-AR subtypes and of a number of functionally different receptor mutants was instrumental to make hypothesis about the potential molecular mechanisms underlying the transition between the inactive ŽR. and active states ŽRU . of the receptor. Our findings highlighted a network of hydrogenbonding interactions among some conserved polar residues forming a ‘‘polar pocket’’ near the cytosol ŽFig. 1. and the arginine of the ErDRY motif. We suggested that this set of interactions constrains the receptor in its ground or inactive state ŽR. by controlling the degree of cytosolic exposure attainable by the arginine of the ErDRY sequence. This hypothesis is supported by the observation that the charge reinforced H-bonding interaction between the conserved aspartate on helix 2 and the arginine of the ErDRY sequence constitutes one of the constraining interactions in the receptor ground state. In contrast, a landmark of the constitutively active receptor mutants is the
breakage of this interaction and the shift of the arginine out of the ‘‘polar pocket’’ ŽFig. 1.. The results of the MD simulations indicate that the motions of the helix bundle triggered by different activating mutations in the a 1-AR subtypes induce the breakage of the interactions within the ‘‘polar pocket’’ and a rearrangement of the cytosolic domains which seem to form a site with docking complementarity with the G protein ŽFig. 1. ŽFanelli et al., 1999.. Our findings suggest that the main role of the arginine of the ErDRY motif is to mediate receptor activation either directly interacting with the G protein heterotrimer or allowing several cationic amino acids in i2 and i3 to attain the right configuration for the formation of a site with docking complementarity with the G protein. This hypothesis is supported by the experimental findings showing that different mutations of R143 can almost entirely inactivate the a 1b -AR ŽScheer et al., 1996.. 5. Inverse agonism and neutral antagonism at the a 1a and a 1b -AR subtypes Both selective and nonselective antagonists for different AR subtypes are widely used in a variety of pathological conditions including hypertension, heart failure, prostate hypertrophy as well as in mental diseases such as depression. Several studies have demonstrated that beta-blockers can behave either as neutral antagonists or inverse agonists at the wild type b 2-AR or at a constitutively active b 2-AR mutant ŽSamama et al., 1993a,b; Chidiac et al., 1994.. On the other hand, inverse agonism at other AR subtypes has been less extensively investigated. It has been previously reported that a small range of alpha-blockers could inhibit the agonist-independent phospholipase C as well as phospholipase D responses mediated by constitutively active mutants of the a 1b -AR ŽLee et al., 1997.. A recent study has demonstrated that some alpha-blockers can inhibit the spontaneous activity of the a 1d -AR subtype ŽGarcıa-Sainz ´ ´ and Torres-Padilla, 1999..
Fig. 2. Constitutively active a 1a -AR and a 1b-AR mutants. D123 and A271 of the a 1a-AR as well as D142 and A293 of the a 1b-AR have been mutated into alanine and glutamic acid, respectively. Receptors expression in COS-7 cells ranged 1.5–2.5 pmolrmg of protein. Inositol phosphate Žw3 HxIP. accumulation was measured in cells expressing the wild type or mutated receptors after incubation in the absence ŽBasal. or presence of 10y4 M epinephrine for 45 min. Results are the mean"S.E of three independent experiments.
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We have recently characterized the pharmacological antagonism, i.e., neutral antagonism or inverse agonism, displayed by a number of alpha-blockers at two a 1-AR subtypes, the a 1a and a 1b-AR ŽRossier et al., 1999.. One strategy to identify inverse agonists is to screen drugs for their ability to inhibit the agonist-independent activity of the constitutively active receptor mutants. Thus, 24 alphablockers differing in their chemical structures were tested for their effect on the basal activity of the constitutively active A271E and A293E mutants expressed in COS-7 cells ŽFig. 3.. All the ligands used in this study, except REC 15r3039, were previously described for their structure, their binding affinity at recombinant as well as native a 1-AR subtypes and some of their pharmacological effects in different tissues ŽMichel et al., 1995; Leonardi et al., 1997; Testa et al., 1997.. Our results show that the vast majority of alpha-blockers displayed inverse agonism as demonstrated by their
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ability to decrease the basal activity of both CAMs. However, the various alpha-blockers differed in their negative efficacy and some of these differences depended on the a 1-AR subtype. Drugs with the highest negative efficacy Ždefined as G 70% inhibition of the basal activity. at both CAMs included WAY 100635, WB 4101, all the tested quinazolines Žprazosin, terazosin, both Žq.- and Žy.cyclazosin, REC 15r2615 and alfuzosin., indoramin, corynanthine, spiperone and AH11110A ŽFig. 3.. For the other drugs, their negative efficacy differed at the two CAMs. The most striking difference concerned some N-arylpiperazines which displayed modest negative efficacy Že.g., 5-methylurapidil, BMY 7378 and REC 15r2869. or neutral antagonism Že.g., REC 15r3039, REC 15r2739 and REC 15r3011. at the A271E mutant. On the other hand, the negative efficacy of these compounds was more pronounced at the A293E resulting in at least 45% inhibition of the receptor-mediated basal activity. For phentolamine,
Fig. 3. Ligand-induced inhibition of the basal inositol phosphate response mediated by constitutively active a 1 -AR mutants. Inositol phosphate Žw3 HxIP. accumulation was measured in COS-7 cells expressing the wild type ŽWT. a 1a -AR and a 1b -AR and their respective mutants A271E and A293E. In cells expressing the receptor mutants, w3 HxIP were measured in the absence ŽBasal. or presence of different ligands at a concentration of 10y5 M Ž10y4 M for REC 15r3039. for 45 min. Mock indicates cells not expressing the receptors. The ligands are grouped according to their structural similarities Žfrom left to right the groups include: N-arylpiperazines; 1,4-dihydropyridines; imidazolines; benzodioxanes and phenylalkylamines; quinazolines; various structures.. Results are the mean"S.E of three to six independent experiments.
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BE 2254 and tamsulosin negative efficacy was also greater at the A293E than at the A271E. In contrast, for Ž S .-Žq.niguldipine negative efficacy was greater at the A293E than at the A271E mutant. To assess whether the effect of the alpha-blockers observed on the CAMs reflected their behaviour at the wild type receptors, a selected number of drugs were tested for their effects in COS-7 cells expressing the wild type a 1a and a 1b -AR subtypes. Overexpression of both a 1-AR subtypes resulted in a small, but measurable increase of agonist-independent accumulation of IP which was greater for the a 1b -AR than for the a 1a-AR. The effect of the alpha-blockers in cells expressing the wild type a 1-AR subtypes displayed several similarities to that observed in cells expressing their CAMs. Altogether, our findings identify a group of N-arylpiperazines as alpha-blockers which display the most striking difference at the two a 1-AR subtypes being inverse agonists with profound negative efficacy at the wild type a 1b -AR, but not at the a 1a-AR. In particular, REC 15r3039, REC 15r2739 and REC 15r3011 are the first alpha-blockers identified so far which do not display inverse agonism at one of the a 1-AR subtypes, namely, the a 1a . A preliminary structure-activity relationship analysis of the ligands suggests that the inhibitory effect observed for different ligands on the constitutive activity of the a 1a -AR and, to a smaller extent, on that of the a 1b -AR is related to the structure of a defined portion of the ligands. Our results suggest that the geometry of the protonated nitrogen atom as well as that of the molecular moiety closest to this nitrogen might be responsible for the functional effect of the ligands tested ŽRossier et al., 1999.. The group of quinazolines including prazosin, terazosin and alfuzosin, which are among the most commonly used alpha-blockers, can display unwanted effects in vivo on the cardiovascular system such as orthostatic hypotension. In contrast, REC 15r2739, REC 15r2869 and REC 15r3011 are characterized by high selectivity for the urogenital tissues and seem to have less pronounced effects on the cardiovascular system in vivo ŽTesta et al., 1997.. In future studies, it will be interesting to identify novel alpha-blockers which are neutral antagonists at one or more a 1-AR subtypes and to assess whether these compounds, including REC 15r3039, have fewer generalized cardiovascular effects as compared to full inverse agonists.
6. Conclusions. The results of our recent studies provide new information on the activation process of the a 1a and a 1b-AR subtypes as well as on the mechanisms of action of drugs acting at these receptors. They might also provide generalities about the molecular mechanisms underlying the activation process of other GPCRs. The structural conservation
among different receptors suggests that the molecular mechanisms proposed for the a 1a - and a 1b-AR subtypes might be generalized to the activation process of other members of the GPCR family. We propose that an interdisciplinary approach combining site-directed mutagenesis, molecular dynamics and thermodynamical analysis of receptor mutants is useful to elucidate the structural-dynamic properties of GPCRs.
Acknowledgements This work was supported by the Fonds National Suisse de la Recherche Scientifique Žgrant 31-51043.97. and by the European Community Žgrant BMH4-CT97-2152..
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