Molecular pharmacology and drug action: structural information casts light on ligand binding

T i P S - J u n e 1989 [118] !

i

~

i

;

]

Volume 10, No. 6 !

~

.

i

i

~

.

-

. . . .

2

t

Molecular pharmacology and drug action: structural information casts light on ligand binding HOW CAN AGONISTS and antagonists be discriminated at a molecular level? This question formed the basis of discussion at a recent Titisee conference*. To even begin to answer this question, the molecular functions of drug receptors must be understood, and the recent cloning of the genes for numerous drug receptors is throwing a light on this problem. It is now clear that receptors belong to structurally related families such as G protein coupled receptors or ligand-gated ion channels. Also, receptor subtype diversity, as revealed by molecular biolog~ has been shown to be very large, larger than ever anticipated. On the basis of this knowledge, participants grappled with questions concernix~g the nature of drug binding sites, how effector molecules are coupled and the role of receptor heterogeneity. In the course of the three-day meeting, other advances in molecular pharmacology were presented, including the first sequence data for a mammalian adenylyl cyclase. Adenylyl cyclase The elusive adenylyl cyclase has finally yielded to cloning technology. A1 Gilman (University of Texas, Dallas) presented his group's recently acquired primary amino acid sequence data on bovine brain adenylyl cyclase. Somewhat surprisingly, its modelled topography places it in the general family of transporter or channel proteins. The sequence contains two similar domains of around 250 amino acids; comparison of these domains with other protein sequences indicates that they may specify nucleotide binding sites. Thus they have been shown to be similar to a cytoplasmic domain present in the four guanylyl cyclases that have so far Continued on p. 208 ~) 1989, Elsevier Science Publishers Ltd. (UK) 0165 - 6147189/$02.00

2~ common topology with seven transmembrane regions and by varying degrees of primary sequence similarity3. Work reported at the conference indicates that agonists and antagonists actually bind within the transmembrane domains. More than 20 receptors from this family have now been cloned. However, 'the list', compiled by Lutz Birnbaumer (Baylor College, Houston), includes at least 70 different receptors. This number is certain to grow as new receptor subtypes continue to be identified. Within the adrenoceptor family, two subtypes of the c~2-receptor have been cloned (Marc Caron, Duke University, Durham). They correspond to the platelet and kidney oc2-adrenoceptors and, while they both have o~2-adrenergic pharmacology, they can be distinguished by their affinities for epinephrine, norepinephrine and oxymetazoline. Proteolytic removal of the large extracellular and intracellular domains from purified ~l-adrenoceptors (Elliot Ross, University of Texas, Dallas) showed that ligands bound within the remaining transmembrane domains. Moreover, a tryptophan residue in the seventh transmembrane segment was identified as a portion of the ligand binding site by photolabelling. In another set of experiments described by Marc Caron, chimeric oc2-~2-adrenoceptors were constructed. Switching of transmembrane domains (TM) indicated that TM7 of these receptors appears to be particularly important in determining ligand binding specificity. Structure--function studies indicate that negatively charged aspartic acid residues within the second and third transmembrane domains are crucial for ligand binding 4"5. These domains have also been identified by various photolabelling experiments 6. The role of these negatively charged residues appears to be in binding (or channelling) positively charged ligands (e.g. adrenergic, rauscarinic) into the centre of the receptor. Nigel Birdsall (National Institute of Medical Research, London) examined the pH dependence of antagG protein-coupled receptor onist binding to m2 (cardiac) mussuperfamily carinic receptors. Most antagonis~s The G protein-coupled 2 family possess a single positive charge of receptors is characterized by a and can act with either of two receptor-borne carboxyl groups *D,-ug Action at the Molecular Level: Di[(presumably two of the conserved [erences Between Agonists and Antagonists, Titisee, FRG, 12-16 April 1989. aspartate residues in TM2 and

continued from front page

been cloned (see Fig. 1). Although the regulatory mechanisms of these enzymes may differ from adenylyl cyclase, their catalytic mechanisms may be assumed to be the same. Furthermore, when compared with protein data banks, the nine highest homology scores were observed with other nucleotide binding proteins. Hydropathy data indicate the protein to have a pair of six membrane-spanning segments separa:cd by a large (43kDa) cytoplasmic loop and one major extracellular loop containing one of the four possible glycosylation sites in the sequence. There is a short cytoplasmic amino-terminal sequence and a long (36kDa) cytoplasmic tail. Thus most of the protein in the Gilman model is located on the cytoplasmic side of the membrane. The proposed transmembrane spans have been modelled as c~helices and several of these are amphipathic. The structure that Gilman has hypothesized for adenylyl cydase bears a surprising resemblance to proteins of markedly different function. Transporters and channels such as the dihydropyridinesensitive Ca 2+ channel and the drug-efflux pump - the P-glycoprotein whose synthesis is enhanced in multidrug-resistant cells 1 - share the feature of large cytoplasmic domains separating sets of six transmembrane spans. P-glycoprotein0 like adenylyl cyclase, is hypothesized to have a short amino-terminal cytoplasmic domain, two sets of six transmembrane spans separated by a large cytoplasmic domain and a large carboxy-terminal cytoplasmic tail; each of the large cytoplasmic domains contains an ATP binding site. These similar topographies do not reflect similar primary amino acid sequences- but do they reflect similar functions? Although apparently unlikely, there may be some evolutionary logic to this question: the cellular slime mold Dictyostelium discoideum exports c A-,MPas an extracellular signal for chemotaxis.

TiPS - J u n e 1989 [Vol. 10]

TM3). It is also possible for two antagonists to bind simultaneously to the receptor with the positive charge of each antagonist interacting with a separate carboxylate residue. In the case of methoctramine, an antagonist witr~ multiple positive charges, its simultaneous interaction with both carboxylate residues was demonstrated. It appears that cardioselective antagonists of differing selectivities interact predominantly with one particular aspartate. This choice is manifest in the magnitude of the apparent pK estimates for protonation of the receptor when the different antagonists bind. The role of the interaction of positively charged residues on the cytoplasmic surface of the receptor with G proteins was suggested by Elliot Ross. He described the photolabeUing of G protein 0c-subunits by the wasp venum mastoparan. This helical peptide presents a face of positively charged residues which can activate the G protein. This face may be akin to a similar motif present in the putative G protein binding domain of this receptor family. G proteincoupling sites on the receptors include three domains on the cytoplasmic side of TM5, TM6 and TM7 (Marc Caron). Together, these results indicate that the transmembrane domains fold around to make a pocket in which the ligand binds. The cytoplasmic side of this structure has a number of contact points for G proteins, thus providing a means of signal transduction. A novel covalent modification of the human ~2-adrenoceptor in which palmitate is attached by a thioester linkage to a cytoplasmically located cysteine residue (C-terminal to TM7) was also described by Marc Caron. This cysteine residue is conserved between various members of the superfamily. Mutating this residue to a glycine results in no incorporation of palmitic acid and also produces receptors that are essentially uncoupled from G protein interaction.

G proteins ~.ine different genes have been so far feuncl to code for G proteins, and the fai~,ily is expected to grow (Gilman, University of Texas). The 0c~y-trimer is necessary for interaction with receptor, but disso-

TiPS - Jtuze 1989 CVol. 101

209

Arguments about the ac&pt&lity of the term ‘partial antagonist’ continued on in the bar . . . . . . while the arguments about the G protein subunit that is the active mediator of hormone action moved towards resolution.

The newly sequenced adenylyl cyclase was revealed to have topographical but not sequence similarity with other known structures including the Ca2+ channel, the drug efflux pump and the Loch Ness monster.

Thegrouppho!oshowsthe participants enjoying clement weather in the Black Forest location of the meeting.

TiPS- June 1989 [Vol. 10]

210 ciation of the subunits is required for activation. In the continuing debate about which subunit is the active mediator of hormonal signal to effector (the variant oc, or the invariant [~y),all participants at the meeting declared themselves '0~chauvinists' (a term coined by Henry Bourne in a recent issue of NatureT). The differing results from the Clapham and Birnbaumer labs on activating the K÷ channel with [3y- and c~-subunits respectively had tentatively been ascribed to contamination of subunit preparations s. However, the differences can now be put down to levels and type of detergent used in the assays (CHAPS vs Lubrol, concentration cut-off point 1 ~ ) ; Lutz Birnbaumer maintains his belief that the major cellular role of [~y is as a "noise suppressor', the dimer acting from a general pool to attenuate actions of all G~s acting in a normal membrane environment. Pete Downes (Smith Klein and French) reported experiments where the relationship of Gp, _~ putative pertussis toxin-insensitive G protein assumed to regulate phospholipase C, to Gs was investigated. [~y-Subunits from other (non-Gp) G proteins were added at varying concentrations to turkey erythrocytes in which phospholipase C had been activated by fluoride. Inhibition of both phospholipase C and adenylyl cyclase was observed over the same concentration range of [3y, indicating that [~¥has similar affinities for Gp and Gs. Various fine-tuning mechanisms of the regulatory G protein cycle were suggested. Alex Levitzki (Hebrew University of Jerusalem) suggested that experimental data gathered to date do not preclude the possible permanent association of G~s with adenylyl cyclase. Birnbaumer pointed out that it was not yet possible to determine which step was controlled by the hormonereceptor-G protein complex- the binding of GTP or the activation of the whole complex. [igand-gated ion channel receptors The ligand-gated ion channel receptors are multimeric protein complexes. In the prototypical example, the neuromuscular nicotinic acetylcholine receptor, the receptor consists of a pentameric

structure in which five structurally related subunits are assembled to generate an integral ion channel. Each subunit is believed to contain four transmembrane domains, of which one, TM2, forms the ion channel lining 9. Thus, unlike the G protein-coupled receptors, the transmembrane domains of the ligand-gated ion channel receptors do not appear to form the ligand binding domain. The coral-derived toxin, lophotoxin, photolabels the 0~-subunit of the Torpedo nicotinic acetylcholine receptor and Tyr190 is labelled in a competitive manner (Palmer Taylor, University of California, San Diego). This, and other work (such as that by Changeux and coworkers1°), has demonstrated that the acetylcholine binding site is very close to two cysteine residues located at positions 192 and 193, in the extracellular region immediately adjacent to the transmembrane domains of the ion channel. An analogous site in the glycine receptor, where a putative disulphide-bonded loop also exists, was proposed by Heinrich Betz (Centre for Molecular Biology, Heidelberg) to be the competitive binding site for strychnine. Interestingly, no equivalent disulphide bond is found in any of the GABAA receptor subunits. Receptor subtypes have also emerged as one of the distinguishing features of studies on the nature of drug action. Ralf Schoepfer (Salk Institute, San Diego) outlined the progress that has been made in linking neuronal nicotinic acetylcholine receptor proteins to their cognate genes. In the chick brain, two receptor subtypes have been identified that share a common structural subunit (designated [3 or non-00 but differ in acetylcholine-binding subunits (00. Both receptor subtypes are insensitive to 0c-bungarotoxin, despite the presence of bungarotoxin binding sites in the brain. Schoepfer described two new receptor subunits that, although members of this receptor superfamily, are only distantly related to both 0~- and non-0~-subunits. Subunit-specific antibodies are capable of immunoprecipitating 0c-bungarotoxin labelled receptors. Peter Schofield (Pacific Biotechnology, Sydney), describing experiments undertaken at the University of Heidelberg, reported

that 13 different GABAA receptor subunits had been cloned. These represented five different families, three of which contained highly related subtypes. Changing oc-subunit combinations appeared to alter the affinity of receptors for ligand, while the presence of the y2-subunit was required to observe the full benzodiazepine pharmacology, including allosteric potentiation of GABA responses and the effects of inverse agonists. By altering the receptor subunit combinations, receptor subtypes are obtained that have pharmacological profiles that match the type I and type II benzodiazepine receptors. In-situ hybridization shows that mRNA encoding the 6subunit is found in various interneurons, suggesting a role in local signal modulation, whereas the Y2subunit mRNA for example is located in a totally different set of neurons. Thus, even these two subunits define distinct GABAA receptor subtypes. All known GABAA receptor heterogeneity derives from separate genes encoding the various subunits; however, novel variants generated by alternative splicing were reported for the glycine receptor (Betz). Betz has also isolated a cDNA encoding an embryonic form of the strychnine-binding subunit of the glycine receptor. The developmental change in levels of adult and embryonic subunit expression correlates with a juvenile insensitivity to strychnine. At the functional level, David Colquhoun (University College, London) described work performed by Claire Newland in which extraordinary GABAA receptor heterogeneity was seen in single channel recordings of ganglionic membrane patches. Either of two interpretations would raise many questions: namely, is this heterogeneity caused by a single channel with great variability or by many different channels? Each of the known GABAA :eceptor subunits (Schofield) and the strychnine-binding subunit of the glycine receptor (Betz) can form homomeric ligand-gated ion channels. In the latter case, Hill coefficients of 1.5-3.0 are seen, indicating positive cooperativity. Both glycine (Betz) and nicotinic acetylcholine receptor (Bob Stroud, University of California, San Francisco) transmembrane

TiPS June 1989 [Vol. 10]

211

-

peptides will also form channels when reconstituted in lipid bilayers. Unfortunately, the channels typically have long open times; the rest of the receptor must therefore be important in providing a means of channel gating. The stoichiometry of receptors in vivo continues to be controversial. Whole receptor iodination combined with individual subunit analysis suggests that the neuronal nicotinic acetylcholine receptor may have a tetrameric structure (Schoepfer). This is in contrast to the pentameric structures of the neuromuscular nicotinic acetylcholine receptor (Stroud) and the glycine receptor (Betz). Many of the issues concerning receptor structure and function will be clarified when crystal structures are resolved. Stroud has obtained nicotinic acetylcholine receptor crystals. However, they refract with poor resolution. Hartmut Michel (Max Planck Institute for Biochemistry, Frankfurt), who outlined the most elegant work on the crystal structure of the photoreaction centre, considered that many membrane proteins would not yield usable crystals. Despite this, a single structure for any member of a receptor superfamily would provide information applicable to other members. Jeff Watkins (University of Bristol) described the rapidly growing pharmacology of the excitatory amino acid (glutamate) receptors, of which multiple subtypes have already been defined (NMDA, kainate and quisqualate). Dozens of labs are attempting to purify and/or clone these receptors. Indeed, some dinner-table conversations planned the content of the first glutamate receptor p a p e r - the feeling being that we already know much about this important receptor class.

EGF receptor The EGF receptor is a single, membrane-spanning polypeptide. Following ligand binding at the extracellular domain, receptors dimerize and the intrinsic protein tyrosine kinase activity within the intracellular domain is activated (see Ref. 11). The transmembrane segment appears to serve only an anchor role. Several hundred mutant receptors (]ossi Schlessinger, Rorer Pharmaceuticals Inc., King of Prussia) and chimeric

receptors (Axel Ullrich, MaxPlanck-Institut f~r Biochemie, Martinsried) have been synthesized to analyse in more detail the molecular requirements for signalling through this receptor system. For example, chimeric EGF-insulin receptors can be synthesized and transported normally in cells. Experiments with these chimeras demonstrate that a ligand for the extracellular domain can activate the intracellular domain of a different receptor, but the source of the intracellular domain dictates whether or not a receptor will be degraded (EGF) or recycled (insulin). Single point mutations can lead to oncogenic transformation of EGF receptors which demonstrate ligand-independent constitutive tyrosine kinase activity. In this regard, the new family of selective tyrosine kinase inhibitors introduced by Levitzki has obvious potential. From the lead compound erbstatin (ICs0 "" 141is) found in Streptomyces by Umezawa and colleagues in 1986, Levitski's group has developed a series of compounds, some of which they have termed tyrphostins, with improved affinity and selectivity for EGF over insulin receptors and other tyrosine kinases.

Steroid hormone receptors The question as to how agonists and antagonists differ at the molecular level was approached most directly during discussion of steroid hormone receptors. Unlike membrane-bound peptide receptors, steroid receptors are localized in the soluble fraction of the cell. Following steroid binding, the complex is activated, binds to specific sequences in the chromatin and initiates transcription and protein synthesis Receptors for all steroid hormones .'r~ organized into three major domairts: a variable anaino-terminal domain; a relatively well-conserved carboxyterminal domain which contains the hormone-binding region; and a well-conserved, cysteine-rich central domain 12. The carboxyterminal domain is also responsible for interaction with the 90 kDa heat-shock protein (hsp90) which dissociates on hormone binding, thereby having a possible indirect effect on nuclear translocation (movement of the complex to the chromatin). The central

Quotes from the meeting

'It does not matter what you find, you must give it a name.' A. Levitzki

'When the news broke that adrenoceptors had a structure similar to rhodopsin it struck the receptor community like a bomb.'

PdcA's'lP

o'r 84E

I

I

"*"-''"I

" ~ - ~ "~ ..,if/,.

+I+

Pierre de Meytes summarized the high-powered meeting.

212 domain, with its two Zn2+-stabi lized 'fingers' and additional hormone-dependent translocation signals, is most likely responsible for DNA binding of the receptor. In the absence of hormone, the DNA binding site of the receptor may be hidden within its tertiary structure; following agonist binding, conformational changes expose this region, allowing binding of the steroid receptor to the hormone-responsive element (HRE), an enhancer region upstream of the genes that are regulated. HREs are palindromic or near palindromic consensus sequences which bind steroid receptors. Since sometimes more than one receptor can bind to a consensus sequence, steroid specificity of gene activation must also involve relative titre of receptors and differences in t r a n s - a c t i v a t i o n . John Katzenellenbogen (University of Illinois, Urbana) reported several differences between agonists and antagonists. In cell-free preparations, differences are apparent in ligand dissociation rates, sensitivities to proteases and thiol reagents, aggregation with hsp90, monoclonal antibody binding and elution from ion-exchange columns; in nuclear preparations, antagonist-receptor complexes have a greater tendency to form

T i P S - June 1989 [Vol. 10]

dimers and are less rapidly processed; and in chromatin binding, antagonist complexes show different binding kinetics. It is not clear, however, at what level these differences are critical, nor whether i n - v i t r o chromatin binding and transfection systems are providing fully accurate models for steroid antagonist action in v i v o . Ullrich Gehring (Institute for Biological Chemistry, Heidelberg) presented evidence that the glucocorticoid receptor from $49 1~nnphoid cells has differential stability when bound to agonist (triamcinolone) or antagonist (RU38486). Following agonist binding, activation by high salt (300mM KCI) or warming causes the heterotetrameric glucocorticoid receptor to dissociate, and a low molecular weight form binds to DNA. Receptor-antagonist complexes activated by salt at low temperatures follow a similar pattern of activation and the low molecular weight form can be shown by footprinting techniques to bind to the same specific sequence in the HRE. However, activation by warming to 28°C or 30°C (closer to physiological reality) caused dissociation of the antagonist from the receptor. Once the ligand has left the activated receptor which is in its 'unfolded' form, it is rapidly

Current awareness •

.

.

.

.

.

Similarities in mode and sites of action of sarafotoxins and endothelins A new peptide-receptor communications pathway linked to the phosphoinositide second messenger system and to the Ca 2+ signal-transduction mechanism has recently been uncovered by the use of snake venom toxins 1, and by the discovery of the mammalian vasoconstrictor peptide endothelin 2. The vasoconstrictorcardiotoxic peptides, the sarafotoxins (SRTXs), which are derived from the naO_'veIsraeli snake A t r a c taspis eingadensis 3,4, bind with high affinity to a novel membranebound receptor in rat atrium and brain, which is coupled to the phosphoinositide (PI) system 1,s.6. The unusually high degree of sequence homology (Fig. 1) between

the SRTXs (SRTX-a, SRTX-b and SRTX-c) 1'3 and the mammalian endothelins 2"7"8 (now called ET-1, ET-2 and ET-3, see Ref. 9), suggests that the latter peptides might be the endogenous ligands for the 'SRTX receptors'. This notion is supported by direct binding 1°'11 and PI hydrolysis data. T h e e n d o t h e l i n / S R T X receptors

The SRTXs exert powerful effects on those activities of smooth muscle and the atrioventricular conducting system that are thought to be attributable to an increase in intraceUular Ca 2+ (Ref. 12) This finding raised the possibility that the mechanism of action of the SRTXs might involve bind-

~) 1989, Elsevier Science Publishers Ltd. (UK) 0165 - 6147/89/$02.00

degraded. Thus in this lymphoid cell system, molecular events following either antagonist or agonist binding coincide to the point of nuclear association, but very little antagonist-receptor complex is available to bind DNA. PETER R. SCHOFIELD AND ALISON

ABBOTT ~

Pacific Biotechno!ogy Ltd, 74 McLachlan Avenue, Rushcutters Bay 2011, NSW, Australia, and *Trends in Pharmacological Sciences.

References

1 Gottesman, M. M. and Pastan, I. (1988) Trends Pharmacol. Sci. 9, 54-58 2 Gilman,A. G. (1987)Annu. Rev. Biochem. 56, 615-650 3 Dohlman, H. G., Caron, M.G. and Let'kowitz, R. ]. (1987) Biochemistry 26, 2657-2664 4 Strader, C. D. et al. (1988.)J. Biol. Chem. 263, 10267-10271 5 Fraser, C. M., Chung, F-Z., Wang, C-D. and Venter, J. C. (1988) Proc. Natl Acad. Sci. USA 85, 5478-5482 6 Strader,C. D. et al. (1987)Proc. Natl Acad. Sci. USA 84, 4384--4388 7 Bourne, H. R. (1989)Nature 337, 504-505 8 Birnbaumer, L. (1987) Trends Pharmacol. Sci. 8, 209-211 9 McCarthy, M. P., Earnest, j. P., Young, E. F., Choe, S. and Stroud, R. M. (1986) Annu. Rev. Neurosci. 9, 383-413 10 Dennis, M. et al. (1988) Biochemistry 27, 2346--2357 11 Ramachandran, J. and Ullrich, A. (1987) Trends Pharmacol. Sci. 8, 28-31 12 Beato, M. (1989) Cell 56, 335-344 TiPS will be publishing a short review on tyrphostins later in the year.

ing to and activation of a specific protein associated with Ca 2+ mobilization. Preliminary studies indicated that SRTXs did not interfere with the binding of the Ca 2+ channel antagonists [3H]nitrendipine or [3H]verapamil to rat atrial membranes, nor did they affect the binding of muscarinic or noradrenergic ligands to their respective receptors in the heart. Thus, the direct effects of SRTXs on the heart could not be related to an interaction with known membrane-bound receptors or Ca 2+ channels associated with Ca 2+ mobilization. In addition, SRTXs induced dose-dependent PI hydrolysis in rat atrial slices at low doses (100 nM) and in the absence of extracellular Ca 2+, this activity was not inhibited by known receptor antagonists or channel blockers. All these findings pointed to the existence of a highly specific receptor-phospholipase C system with which the SRTXs