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Signalling in the β-adrenergic receptor system
Proceedings, XIVth International Symposiumon Medicinal Chemistry F. Awouters (Editor) 9 1997 Elsevier Science B.V. All rights reserved.
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Signalling in the ~-adrenergic receptor system C. Krasel and M. J. Lohse Institute of Pharmacology, University of Wtirzburg, Versbacher Strasse 9, D-97078 Wtirzburg, Germany 1. ABSTRACT The mechanisms of activation and inactivation of G-protein-coupled receptors are beginning to be unravelled at the molecular level. Using mostly the I]-adrenergic receptor system and the light receptor, rhodopsin, as prototypical models, several critical steps in these processes have been identified. Activation of receptors is initiated by agonist binding. The binding of the l-selective agonist (-)isoproterenol has been demonstrated to occur via at least 4 attachment points between the ligand and side chains in the receptors' transmembrane helices, which correspond to the secondary amino group, the two catechol OH-groups and the stereospecific 13-OH group in isoproterenol. We propose that the interaction of the ~-OH group with the 6th transmembrane helix may be relevant for both stereoselective agonist recognition and receptor activation. Agonist-induced movement of this helix may then be transmitted to the cytosolic loops of the receptor, which interact with the G-protein. Inactivation of receptors can be triggered by various processes, most notably by phosphorylation of the receptors by different protein kinases. These include (1) the effector kinases, i.e. protein kinase A (PKA) and protein kinase C (PKC), and (2) specific kinases termed 13-adrenergic receptor kinases (I]ARK). Recent data indicate that these modes are, in addition, interconnected. Thus, I3ARK has to translocate from the cytosol to the membrane in order to phosphorylate receptors. For this purpose it utilizes three membrane anchors: the activated receptor, the G-protein 13y-subunit complex, and certain membrane lipids. Signalling is also regulated at the G-protein level. We have recently identified phosducin and a phosducin-like protein as ubiquitous G-protein regulators. These proteins bind to the G-protein 137-complex and thereby disrupt the signalling process. The mode of binding is distinct from the mode of lARK binding to the G-protein ~y-complex and appears to have different structural requirements. Again, this regulatory mechanism is subject to control by a protein kinase: PKA can phosphorylate and thereby inactivate phosducin. The functional relevance of these regulatory mechanisms can be shown in two ways: (1) Modulation of the expression of such regulatory proteins results in distinct alterations of transmembrane signalling, and (2) in certain diseases, such as heart failure, the expression of these proteins is altered and this results in modulations of receptor function which may be relevant for disease induction and progression.
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2. LIGAND BINDING TO ~-ADRENERGIC RECEPTORS The adrenergic receptor family consists of three subfamilies with three members each: the a 1-receptors coupled to stimulation of phospholipase C, the a2-receptors coupled to inhibition of adenylyl cyclase, and the [3-receptors coupled to stimulation of adenylyl cyclase. Among the 13-adrenergic receptors, a 131-, a 132-, and a I33-subtype can be distinguished. In addition, there are at least two avian adrenergic receptors which display [3-like ligand selectivities but differ in other properties [ 1] and therefore will not be discussed here. All 13-adrenergic receptors are members of the large superfamily of G-protein-coupled receptors and share their common molecular architecture: an extracellular amino terminus with several sites for N-linked glycosylation, seven hydrophobic domains which traverse the plasma membrane presumably in an a-helical conformation and form the binding pocket for the ligand, and an intracellular carboxy terminus which contains one or more conserved cysteine residues. In the [32 subtype, this residue is palmitoylated, thereby forming an additional intracellular loop (Figure 1). This model is supported by a multitude of experimental findings [2].
~ I ~~~ Intracellular
~
NH 2
~
Extracellular
. . . . COOH
Figure 1: The topology of the 132-adrenergic receptor in the membrane (indicated by the hatched rectangle). N-glycosylation sites are marked by Ys. Black circles denote [3ARK phosphorylation sites. Other receptor kinases may phosphorylate additional sites (diamonds). The protein kinase A phosphorylation site in the third intracellular loop is delineated by the squares and marked with "PKA". Another minor site for the same kinase is located in the C terminus, shortly behind the palmitoylated cysteine. Upon agonist binding, all 13-adrenergic receptors activate membrane-bound adenylyl cyclases via cholera-toxin-sensitive G-proteins, Gs. In addition, activation of the 132-adrenergic receptor has been reported to stimulate Na-H exchange via a Gs-independent mechanism [3]. The mechanism of agonist binding has been most thoroughly studied for the 132-adrenergic receptor. When it was cloned, its amino acid sequence immediately suggested a close topological similarity to the rhodopsins [4]. A conformation with seven transmembrane helices
319 had already been established for bacteriorhodopsin by electron diffraction studies of twodimensional crystals [5]. Soon thereafter, it was demonstrated that mutant receptors lost their ability to bind ligands when the hydrophobic core was affected [6]. These findings were confirmed by showing that [~-adrenergic receptor purified from turkey erythrocytes retained its ligand-binding properties even after extensive proteolytic digestion which left only the membrane-protected core intact [7]. Mutational analysis indicated that the residues involved in agonist binding are Asp-113 in transmembrane helix III (which probably acts as a counterion for the amino group in the catecholamines) and Ser-204 and Ser-207 in helix V (which are thought to form hydrogen bonds with the catechol OH-groups). From a model based on the structure of bacteriorhodopsin it was proposed that the ~l-OH-group would interact with Ser165 in helix IV [8]; however, very recently it was shown that mutation of this serine to alanine did not have any effect on (-)-isoproterenol binding, while a mutant in which Asn293 was changed to leucine displayed a tenfold higher KD for the agonist [9]. Furthermore, the stereoselectivity of this mutant was impaired since (+)-isproterenol bound with only a sixfold lower affinity whereas in both the wild type and the Ser165Ala mutant it bound with almost 40fold lower affinity. Thus, it was suggested that the ~-OH group interacts with Asn293 which is conserved in all ~-adrenergic receptors. (This binding mode had already been suggested previously [10].) A model of the binding pocket with ligand is shown in Figure 2.
,
1293
Ser207
q
Asp113")
Figure 2: Model of isoproterenol binding to the 132-adrenergic receptor, based on the structure of bacteriorhodopsin [11 ]. Helices are shown as ribbons and marked with roman numerals. The interacting amino acids are shown. Modified from [9].
320 3. H O W DOES A G O N I S T BINDING LEAD TO G - P R O T E I N A C T I V A T I O N ? Once the 132-adrenergic receptor had been cloned, its G-protein activating sites were localized fairly quickly to the second, third and fourth intracellular loop [12]. These findings were supported by competition studies with synthetic peptides [13]. However, it remains a great challenge to find out how exactly binding of an agonist to a G-protein-coupled receptor is propagated through the receptor molecule and leads to activation of the G-protein. Structure elucidation has been hampered by the fact that it is extremely difficult to crystallize membrane proteins. So far, a projection structure of bovine rhodopsin at a resolution of 9 ,~ (the first direct structural view of any G-protein-coupled receptor at all) [14], a three-dimensional lowresolution structure of bovine rhodopsin [ 15] and another projection structure of frog rhodopsin at a resolution of 6 ,~ [16] have been determined, all by cryo-electron microscopy of twodimensional crystals. In the absence of high-resolution structural data, researchers have turned to mutagenesis experiments and modeling to learn more about the molecular mechanisms of G-protein activation by receptors. The GRAP database which covers mutation data of "rhodopsin-like" receptors (this includes most receptors) lists more than 2000 mutants published between 1987 and mid-1995 [17]. From this huge amount of data and an analysis of over 200 receptor sequences a model of the arrangement of the seven helices was constructed [18]. While this model could not identify whether the seven helices were arranged clockwise or counterclockwise when viewed from the "outside", mutational data indicate [19] that helical packing is indeed analogous to that found in bacteriorhodopsin. To supplement this fairly reliable model with high-resolution data, researchers have mostly applied molecular dynamics or simulated annealing to a rough model that is usually built by hand; recently the whole process has even been automated [20]. Most models are only concerned with the binding pocket. Recently, the problem of loop conformation has been approacheded by doing NMR studies of loop peptides at low temperature [21-24]. Whether the resulting structures are applicable in the context of the whole protein is not known. The process of receptor activation has been modeled mathematically in various ways [25]. These models usually assume two different states of a receptor, active and inactive. Early data from binding experiments indicate that in the absence of GTP the active receptor conformation forms a "ternary complex" of agonist, receptor and G-protein. According to these models, agonist induce or stabilize the active conformation while antagonists do not. Biochemically, receptor activation has been most thoroughly researched for rhodopsin, the light-activated G-protein-coupled receptor. Rhodopsin has its "antagonist", the chromophore 11-cis-retinal, covalently bound to Lys296 in the seventh transmembrane helix via a protonated Schiff base. The absorption characteristics of this retinal are very sensitive against changes in its environment, allowing even slight conformational changes in the protein to be monitored spectroscopically. Upon photon absorption, the chromophore isomerizes to all-trans retinal which now acts as an agonist. As a result the proton on the Schiff base moves on to Glu 113, resulting in the break of an ionic interaction between this glutamate and Lys296. In addition, another proton from the cytosol gets bound to Glu134 in the third intracellular loop. The resulting state, called metarhodopsin II, is able to activate the G-protein transducin. A mechanism has been suggested based on kinetic measurements that proposes a sequential activation of the t~ subunit via a GDP-bound, a nucleotide-free and a GTP-bound state [26]. (A
321 three-step mechanism for G-protein activation had been proposed previously, based on more general considerations [27].) In constrast to the allosteric model generally favoured by pharmacologists (see above) this model involves several receptor-G-protein complexes that can be experimentally distinguished from each other. However, it still does not suggest an intramolecular mechanism which would transfer information between the binding site and the cytoplasmic surface. Both the C- and the N-terminal part of the third intracellular loop had been identified early as important determinants of coupling to G-proteins (see beginning of chapter). A stunning observation was made when alanine 293 in the CqB-adrenergic receptor (where it is located at the boundary between the third intracellular loop and transmembrane helix 6; see Figure 3A) was mutated. Replacement with any other amino acid lead to constitutive activation of the receptor [28]. Thus it was proposed that the resting state of receptors is conformationally constrained and becomes more loose upon agonist binding. A similar mutation was identified later in the 132-adrenergic receptor [29].
A
A I
Figure 3: A. Membrane topology of a G-protein-coupled receptor pointing out the location of the DRY motif, the A in the third intracellular loop and the NPXXY motif. B. Low-resolution structure of a G-protein coupled receptor, side view. Helices are numbered. The arginine residue of the DRY motif is shown in its two conformations, switching from the binding pocket (black box) to the cytoplasmic surface (at the bottom). A few other residues that are presumed to be important for this activation mechanism are also shown. Modified from [30]. Based on comparative receptor modelling it was suggested that an arginine residue in the second intracellular loop that is located in the so-called DRY motif and conserved in all Gprotein-coupled receptors may play an important role in transducing the signal from the binding pocket to the intracellular surface (Figure 3) [30]. This arginine residue has been shown to be essential for G-protein coupling to rhodopsin, the ml muscarinic acetylcholine receptor and the angiotensin II receptor. In the resting state, the arginine side chain was suggested to reach out into the polar pocket; upon agonist binding, the side chain flips down to point to the cytosolic surface. In the context of this model, other important residues discussed were a conserved leucine in the second transmembrane helix and the asparagine and tyrosine residues of the
322 NPXXY motif at the cytoplasmic end of the seventh transmembrane helix. In agreement with this model, mutation of the conserved Y of NPXXY disturbs receptor-G-protein coupling [31, 32]. Subsequently, Scheer et al. suggested that in the unoccupied state, these residues would form a network of hydrogen bonds at the bottom of the binding pocket [33]. In an activated receptor, this network would be disturbed. This fits nicely to the work with the constitutively active mutants where the inactive receptor is proposed to be in a more constrained conformation than the agonist-activated one. During this process, the acidic residue in front of the arginine (D in the DRY motif) is supposed to be protonated, a process that has been demonstrated to occur during the activation of rhodopsin, as outlined above [34]. Charge neutralization by mutation at this point leads often, but not always, to constitutive receptor activation [35-37]. Certain mutations in transmembrane helices 5 and 6 also lead to constitutive activation of a l adrenergic receptors [38]. It is believed that these - rather hydrophobic - residues modulate interhelical interactions between the fifth and the sixth transmembrane segment. The importance of helix interactions in this area is further underscored by the observation that the introduction of a zinc-binding site into helix 6 and another helix leads to a zinc-dependent antagonism in both the tachykinin NK-1 receptor [39] (where the engineered zinc-binding site lies between helices 5 and 6) and rhodopsin [40] (where it lies between helices 3 and 6). In both cases, zinc coordination by histidine residues presumably hinders the movement of helix 6 relative to the rest of the receptor.
~q,
.?.
e\
cZ:J 9
"J
./9
,
,
~'
g
Figure 4: Possible receptor - G-protein interaction. At the top the ~2adrenergic receptor model already presented in Figure 2, below the Gi-protein heterotrimer (with the 7 subunit in dark) in two different orientations. The picture underestimates the receptor size by a factor of approximately two since both extra- and intracellular loops are missing from the model.
323
With the structural elucidation of G-protein c~ subunits in various states of activation [41-46] and heterotrimers [47, 48] the question has arisen where exactly the intracellular loops of Gprotein-coupled receptors contact the surface of G-protein subunits. Two possible modes of orientation of the G-protein heterotrimer to the receptor have been proposed [49] and are shown schematically in Figure 4. Sequence homology considerations suggest a relative orientation as shown on the right [50]. 4. DESENSITIZATION BY R E C E P T O R P H O S P H O R Y L A T I O N
Upon prolonged stimulation of 132-adrenergic receptors with agonist, the cAMP response becomes blunted. This phenomenon is called desensitization and is the result of a variety of processes (reviewed in depth in [51]). This review will focus on short-term desensitization which takes place in seconds to minutes within stimulation and is the result of receptor phosphorylation by various kinases. At low physiological agonist concentrations, the 132-adrenergic receptor is preferentially phosphorylated by protein kinase A which has been activated by cAMP production. Phosphorylation occurs at a consensus site in the third intracellular loop (see Figure 1) and decreases coupling efficiency between the receptor and the stimulatory G-protein Gs by up to 60%. The receptor is a substrate regardless of its activation state; stimulation of different Gscoupled receptors may lead to phosphorylation of other types of receptors. The 131-adrenergic receptor is less sensitive to protein kinase A-mediated desensitization and the 133-adrenergic receptor is completely resistant. The ]]2-adrenergic receptor may also be phosphorylated by protein kinase C which phosphorylates the same site. At high agonist concentrations, 131- or 132-adrenergic receptors become substrates for a family of kinases called G-protein-coupled receptor kinases (GRKs) [52-54], while the [33adrenergic receptor is not a substrate for this kinase family. Of the GRKs, rhodopsin kinase (which occurs only in the retina) and the two 13-adrenergic receptor kinases (13ARK) have received most attention. Phosphorylation of adrenergic receptors takes place at serines and threonines in the C-terminus which must be surrounded by acidic amino acids (Figure 1). Phosphorylated receptors will then bind with high affinity an uncoupling protein, called 13arrestin. [3-arrestin competes with Gs for the receptor and thereby causes uncoupling between receptors and their G-proteins. Intriguingly, ]3ARK usually occurs in the soluble fraction of cells and moves to the membrane only to phosphorylate activated receptors. This translocation is mediated by free Gprotein 137-subunits [55] and phosphatidylinositol-4,5-bisphosphate binding [56, 57]. It remains somewhat puzzling why most of the ]3ARK seems to localize to the endoplasmic reticulum when stimulated cells are investigated by immunofluorescence [58]. There are no known G-protein-coupled receptors in intracellular organelles. However, an integral membrane protein has been localized to microsomal membranes which is able to bind ]]ARK with high affinity [59]. It is also possible that ]3ARK may phosphorylate yet unknown proteins. Recently it was demonstrated that I3ARK can act as a substrate for protein kinase C [60, 61 ]. PKC-phosphorylated 13ARK will phosphorylate rhodopsin or 132-adrenergic receptor much faster, but not to a higher stoichometry. However, soluble substrates are actually phosphorylated less efficiently which suggests that phosphorylated lARK might have a higher affinity for membranes. Consistent with this finding, a soluble fusion protein which contained the C-terminus of ~ARK comprising both the phospholipid- and the G137-binding domain could
324
be phosphorylated by protein kinase C in vitro [61]. The activation of IgARK by protein kinaseC adds an additional level of complexity to short-term desensitization since desensitization is now also subject to crosstalk between various signal transduction pathways. 5. REGULATION OF G-PROTEIN ACTIVITY Heterotrimeric G-proteins do not only transduce signals from transmembrane receptors to their effectors, they also play important roles in vesicular trafficking [62]. In recent years, an increasing number of proteins have been characterized that are able to interact with G-proteins and regulate their activity [63]. It is yet unknown whether any of these proteins may interfere with signal transduction between [3-adrenergic receptors and adenylyl cyclase. However, it has been shown for one protein, phosducin, that overexpression in A431 cells affected cAMP response to 132-adrenergic agonists [64]. Phosducin is an ubiquitiously expressed protein (with the highest expression in the retina) [65, 66] which can bind G-protein 137-subunits with high affinity [67]. This interaction restricts the availability of 13y-subunits for other proteins, such as o~-subunits [68] or lARK [69]. Phosducin can be phosphorylated by protein kinase A which results in a loss of its GI3y-binding properties [65, 69]. Whether phosducin also interacts with G-protein ~-subunits is a matter of debate. Overexpression of phosducin results in complex effects on adenylyl cyclase activation by the 132-adrenergic receptor [64]. The maximal cAMP production in phosducin-overexpressing A431 cells is approximately 40% of that in wild-type cells, indicating that sequestration of GI3y subunits by phosducin blunts the signal transduction through Gs. This observation is consistent with data obtained in vitro where phosducin inhibits isoproterenol-stimulated adenylyl cyclase activity in A431 membranes [65]. However, during the first minute of stimulation, cAMP production in phosducin-overexpressing cells is increased compared to the wild type. This is due to the competition of phosducin with [3ARK for G-protein Igy subunits which results in impaired lARK activation. This is supported by the observation that desensitization is lower in membranes prepared from phosducin-overexpressing cells. Addition of PKI to permeabilized cells augmented the phosducin effects, showing that only part of the overexpressed phosducin was phosphorylated by protein kinase A. Phosducin-like protein, which was recently cloned from rat brain [70], is 65% homologous to phosducin. It is overexpressed in the brains of ethanol-treated rats [70]; the physiological significance of this observation is unclear. Recombinant phosducin-like protein does also bind to G-protein 137-subunits in vitro, although with a slightly lower affinity than phosducin [71 ]. CONCLUSION Signalling via G-protein-coupled receptors is one of the key processes regulating the activity of cells, their growth and their differentiation. This process gains an increasingly complex appearance because not only the signalling proteins themselves, but also other intracellular proteins seem to be essential components of an entire "receptor system". On the other hand, the mechanisms of signal initiation and transmission as well as those leading to signal termination and to desensitization are beginning to be unravelled at the molecular level. Undoubtedly, future research will be directed at the further elucidation of these molecular structures and mechanisms, and such data will not only help to understand one of the fundamental biological
325
processes but will also become instrumental in defining its pathology and in developing means of pharmacological interference. ACKNOWLEDGEMENTS
Figures 2 and 4 were prepared with MOLSCRIPT [72] and Raster3D [73, 74]. We thank Dr. Steven Sprang for the coordinates of the Gi heterotrimer and Stefan Danner for critical reading of the manuscript. Research in the authors' laboratory is supported by grants from the Deutsche Forschungsgemeinschaft, the Bundesministerium ftir Bildung und Forschung, the European Commission, and the Fonds der Chemischen Industrie. REFERENCES
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Signalling in the ~-adrenergic receptor system C. Krasel and M. J. Lohse Institute of Pharmacology, University of Wtirzburg, Versbacher Strasse 9, D-97078 Wtirzburg, Germany 1. ABSTRACT The mechanisms of activation and inactivation of G-protein-coupled receptors are beginning to be unravelled at the molecular level. Using mostly the I]-adrenergic receptor system and the light receptor, rhodopsin, as prototypical models, several critical steps in these processes have been identified. Activation of receptors is initiated by agonist binding. The binding of the l-selective agonist (-)isoproterenol has been demonstrated to occur via at least 4 attachment points between the ligand and side chains in the receptors' transmembrane helices, which correspond to the secondary amino group, the two catechol OH-groups and the stereospecific 13-OH group in isoproterenol. We propose that the interaction of the ~-OH group with the 6th transmembrane helix may be relevant for both stereoselective agonist recognition and receptor activation. Agonist-induced movement of this helix may then be transmitted to the cytosolic loops of the receptor, which interact with the G-protein. Inactivation of receptors can be triggered by various processes, most notably by phosphorylation of the receptors by different protein kinases. These include (1) the effector kinases, i.e. protein kinase A (PKA) and protein kinase C (PKC), and (2) specific kinases termed 13-adrenergic receptor kinases (I]ARK). Recent data indicate that these modes are, in addition, interconnected. Thus, I3ARK has to translocate from the cytosol to the membrane in order to phosphorylate receptors. For this purpose it utilizes three membrane anchors: the activated receptor, the G-protein 13y-subunit complex, and certain membrane lipids. Signalling is also regulated at the G-protein level. We have recently identified phosducin and a phosducin-like protein as ubiquitous G-protein regulators. These proteins bind to the G-protein 137-complex and thereby disrupt the signalling process. The mode of binding is distinct from the mode of lARK binding to the G-protein ~y-complex and appears to have different structural requirements. Again, this regulatory mechanism is subject to control by a protein kinase: PKA can phosphorylate and thereby inactivate phosducin. The functional relevance of these regulatory mechanisms can be shown in two ways: (1) Modulation of the expression of such regulatory proteins results in distinct alterations of transmembrane signalling, and (2) in certain diseases, such as heart failure, the expression of these proteins is altered and this results in modulations of receptor function which may be relevant for disease induction and progression.
318
2. LIGAND BINDING TO ~-ADRENERGIC RECEPTORS The adrenergic receptor family consists of three subfamilies with three members each: the a 1-receptors coupled to stimulation of phospholipase C, the a2-receptors coupled to inhibition of adenylyl cyclase, and the [3-receptors coupled to stimulation of adenylyl cyclase. Among the 13-adrenergic receptors, a 131-, a 132-, and a I33-subtype can be distinguished. In addition, there are at least two avian adrenergic receptors which display [3-like ligand selectivities but differ in other properties [ 1] and therefore will not be discussed here. All 13-adrenergic receptors are members of the large superfamily of G-protein-coupled receptors and share their common molecular architecture: an extracellular amino terminus with several sites for N-linked glycosylation, seven hydrophobic domains which traverse the plasma membrane presumably in an a-helical conformation and form the binding pocket for the ligand, and an intracellular carboxy terminus which contains one or more conserved cysteine residues. In the [32 subtype, this residue is palmitoylated, thereby forming an additional intracellular loop (Figure 1). This model is supported by a multitude of experimental findings [2].
~ I ~~~ Intracellular
~
NH 2
~
Extracellular
. . . . COOH
Figure 1: The topology of the 132-adrenergic receptor in the membrane (indicated by the hatched rectangle). N-glycosylation sites are marked by Ys. Black circles denote [3ARK phosphorylation sites. Other receptor kinases may phosphorylate additional sites (diamonds). The protein kinase A phosphorylation site in the third intracellular loop is delineated by the squares and marked with "PKA". Another minor site for the same kinase is located in the C terminus, shortly behind the palmitoylated cysteine. Upon agonist binding, all 13-adrenergic receptors activate membrane-bound adenylyl cyclases via cholera-toxin-sensitive G-proteins, Gs. In addition, activation of the 132-adrenergic receptor has been reported to stimulate Na-H exchange via a Gs-independent mechanism [3]. The mechanism of agonist binding has been most thoroughly studied for the 132-adrenergic receptor. When it was cloned, its amino acid sequence immediately suggested a close topological similarity to the rhodopsins [4]. A conformation with seven transmembrane helices
319 had already been established for bacteriorhodopsin by electron diffraction studies of twodimensional crystals [5]. Soon thereafter, it was demonstrated that mutant receptors lost their ability to bind ligands when the hydrophobic core was affected [6]. These findings were confirmed by showing that [~-adrenergic receptor purified from turkey erythrocytes retained its ligand-binding properties even after extensive proteolytic digestion which left only the membrane-protected core intact [7]. Mutational analysis indicated that the residues involved in agonist binding are Asp-113 in transmembrane helix III (which probably acts as a counterion for the amino group in the catecholamines) and Ser-204 and Ser-207 in helix V (which are thought to form hydrogen bonds with the catechol OH-groups). From a model based on the structure of bacteriorhodopsin it was proposed that the ~l-OH-group would interact with Ser165 in helix IV [8]; however, very recently it was shown that mutation of this serine to alanine did not have any effect on (-)-isoproterenol binding, while a mutant in which Asn293 was changed to leucine displayed a tenfold higher KD for the agonist [9]. Furthermore, the stereoselectivity of this mutant was impaired since (+)-isproterenol bound with only a sixfold lower affinity whereas in both the wild type and the Ser165Ala mutant it bound with almost 40fold lower affinity. Thus, it was suggested that the ~-OH group interacts with Asn293 which is conserved in all ~-adrenergic receptors. (This binding mode had already been suggested previously [10].) A model of the binding pocket with ligand is shown in Figure 2.
,
1293
Ser207
q
Asp113")
Figure 2: Model of isoproterenol binding to the 132-adrenergic receptor, based on the structure of bacteriorhodopsin [11 ]. Helices are shown as ribbons and marked with roman numerals. The interacting amino acids are shown. Modified from [9].
320 3. H O W DOES A G O N I S T BINDING LEAD TO G - P R O T E I N A C T I V A T I O N ? Once the 132-adrenergic receptor had been cloned, its G-protein activating sites were localized fairly quickly to the second, third and fourth intracellular loop [12]. These findings were supported by competition studies with synthetic peptides [13]. However, it remains a great challenge to find out how exactly binding of an agonist to a G-protein-coupled receptor is propagated through the receptor molecule and leads to activation of the G-protein. Structure elucidation has been hampered by the fact that it is extremely difficult to crystallize membrane proteins. So far, a projection structure of bovine rhodopsin at a resolution of 9 ,~ (the first direct structural view of any G-protein-coupled receptor at all) [14], a three-dimensional lowresolution structure of bovine rhodopsin [ 15] and another projection structure of frog rhodopsin at a resolution of 6 ,~ [16] have been determined, all by cryo-electron microscopy of twodimensional crystals. In the absence of high-resolution structural data, researchers have turned to mutagenesis experiments and modeling to learn more about the molecular mechanisms of G-protein activation by receptors. The GRAP database which covers mutation data of "rhodopsin-like" receptors (this includes most receptors) lists more than 2000 mutants published between 1987 and mid-1995 [17]. From this huge amount of data and an analysis of over 200 receptor sequences a model of the arrangement of the seven helices was constructed [18]. While this model could not identify whether the seven helices were arranged clockwise or counterclockwise when viewed from the "outside", mutational data indicate [19] that helical packing is indeed analogous to that found in bacteriorhodopsin. To supplement this fairly reliable model with high-resolution data, researchers have mostly applied molecular dynamics or simulated annealing to a rough model that is usually built by hand; recently the whole process has even been automated [20]. Most models are only concerned with the binding pocket. Recently, the problem of loop conformation has been approacheded by doing NMR studies of loop peptides at low temperature [21-24]. Whether the resulting structures are applicable in the context of the whole protein is not known. The process of receptor activation has been modeled mathematically in various ways [25]. These models usually assume two different states of a receptor, active and inactive. Early data from binding experiments indicate that in the absence of GTP the active receptor conformation forms a "ternary complex" of agonist, receptor and G-protein. According to these models, agonist induce or stabilize the active conformation while antagonists do not. Biochemically, receptor activation has been most thoroughly researched for rhodopsin, the light-activated G-protein-coupled receptor. Rhodopsin has its "antagonist", the chromophore 11-cis-retinal, covalently bound to Lys296 in the seventh transmembrane helix via a protonated Schiff base. The absorption characteristics of this retinal are very sensitive against changes in its environment, allowing even slight conformational changes in the protein to be monitored spectroscopically. Upon photon absorption, the chromophore isomerizes to all-trans retinal which now acts as an agonist. As a result the proton on the Schiff base moves on to Glu 113, resulting in the break of an ionic interaction between this glutamate and Lys296. In addition, another proton from the cytosol gets bound to Glu134 in the third intracellular loop. The resulting state, called metarhodopsin II, is able to activate the G-protein transducin. A mechanism has been suggested based on kinetic measurements that proposes a sequential activation of the t~ subunit via a GDP-bound, a nucleotide-free and a GTP-bound state [26]. (A
321 three-step mechanism for G-protein activation had been proposed previously, based on more general considerations [27].) In constrast to the allosteric model generally favoured by pharmacologists (see above) this model involves several receptor-G-protein complexes that can be experimentally distinguished from each other. However, it still does not suggest an intramolecular mechanism which would transfer information between the binding site and the cytoplasmic surface. Both the C- and the N-terminal part of the third intracellular loop had been identified early as important determinants of coupling to G-proteins (see beginning of chapter). A stunning observation was made when alanine 293 in the CqB-adrenergic receptor (where it is located at the boundary between the third intracellular loop and transmembrane helix 6; see Figure 3A) was mutated. Replacement with any other amino acid lead to constitutive activation of the receptor [28]. Thus it was proposed that the resting state of receptors is conformationally constrained and becomes more loose upon agonist binding. A similar mutation was identified later in the 132-adrenergic receptor [29].
A
A I
Figure 3: A. Membrane topology of a G-protein-coupled receptor pointing out the location of the DRY motif, the A in the third intracellular loop and the NPXXY motif. B. Low-resolution structure of a G-protein coupled receptor, side view. Helices are numbered. The arginine residue of the DRY motif is shown in its two conformations, switching from the binding pocket (black box) to the cytoplasmic surface (at the bottom). A few other residues that are presumed to be important for this activation mechanism are also shown. Modified from [30]. Based on comparative receptor modelling it was suggested that an arginine residue in the second intracellular loop that is located in the so-called DRY motif and conserved in all Gprotein-coupled receptors may play an important role in transducing the signal from the binding pocket to the intracellular surface (Figure 3) [30]. This arginine residue has been shown to be essential for G-protein coupling to rhodopsin, the ml muscarinic acetylcholine receptor and the angiotensin II receptor. In the resting state, the arginine side chain was suggested to reach out into the polar pocket; upon agonist binding, the side chain flips down to point to the cytosolic surface. In the context of this model, other important residues discussed were a conserved leucine in the second transmembrane helix and the asparagine and tyrosine residues of the
322 NPXXY motif at the cytoplasmic end of the seventh transmembrane helix. In agreement with this model, mutation of the conserved Y of NPXXY disturbs receptor-G-protein coupling [31, 32]. Subsequently, Scheer et al. suggested that in the unoccupied state, these residues would form a network of hydrogen bonds at the bottom of the binding pocket [33]. In an activated receptor, this network would be disturbed. This fits nicely to the work with the constitutively active mutants where the inactive receptor is proposed to be in a more constrained conformation than the agonist-activated one. During this process, the acidic residue in front of the arginine (D in the DRY motif) is supposed to be protonated, a process that has been demonstrated to occur during the activation of rhodopsin, as outlined above [34]. Charge neutralization by mutation at this point leads often, but not always, to constitutive receptor activation [35-37]. Certain mutations in transmembrane helices 5 and 6 also lead to constitutive activation of a l adrenergic receptors [38]. It is believed that these - rather hydrophobic - residues modulate interhelical interactions between the fifth and the sixth transmembrane segment. The importance of helix interactions in this area is further underscored by the observation that the introduction of a zinc-binding site into helix 6 and another helix leads to a zinc-dependent antagonism in both the tachykinin NK-1 receptor [39] (where the engineered zinc-binding site lies between helices 5 and 6) and rhodopsin [40] (where it lies between helices 3 and 6). In both cases, zinc coordination by histidine residues presumably hinders the movement of helix 6 relative to the rest of the receptor.
~q,
.?.
e\
cZ:J 9
"J
./9
,
,
~'
g
Figure 4: Possible receptor - G-protein interaction. At the top the ~2adrenergic receptor model already presented in Figure 2, below the Gi-protein heterotrimer (with the 7 subunit in dark) in two different orientations. The picture underestimates the receptor size by a factor of approximately two since both extra- and intracellular loops are missing from the model.
323
With the structural elucidation of G-protein c~ subunits in various states of activation [41-46] and heterotrimers [47, 48] the question has arisen where exactly the intracellular loops of Gprotein-coupled receptors contact the surface of G-protein subunits. Two possible modes of orientation of the G-protein heterotrimer to the receptor have been proposed [49] and are shown schematically in Figure 4. Sequence homology considerations suggest a relative orientation as shown on the right [50]. 4. DESENSITIZATION BY R E C E P T O R P H O S P H O R Y L A T I O N
Upon prolonged stimulation of 132-adrenergic receptors with agonist, the cAMP response becomes blunted. This phenomenon is called desensitization and is the result of a variety of processes (reviewed in depth in [51]). This review will focus on short-term desensitization which takes place in seconds to minutes within stimulation and is the result of receptor phosphorylation by various kinases. At low physiological agonist concentrations, the 132-adrenergic receptor is preferentially phosphorylated by protein kinase A which has been activated by cAMP production. Phosphorylation occurs at a consensus site in the third intracellular loop (see Figure 1) and decreases coupling efficiency between the receptor and the stimulatory G-protein Gs by up to 60%. The receptor is a substrate regardless of its activation state; stimulation of different Gscoupled receptors may lead to phosphorylation of other types of receptors. The 131-adrenergic receptor is less sensitive to protein kinase A-mediated desensitization and the 133-adrenergic receptor is completely resistant. The ]]2-adrenergic receptor may also be phosphorylated by protein kinase C which phosphorylates the same site. At high agonist concentrations, 131- or 132-adrenergic receptors become substrates for a family of kinases called G-protein-coupled receptor kinases (GRKs) [52-54], while the [33adrenergic receptor is not a substrate for this kinase family. Of the GRKs, rhodopsin kinase (which occurs only in the retina) and the two 13-adrenergic receptor kinases (13ARK) have received most attention. Phosphorylation of adrenergic receptors takes place at serines and threonines in the C-terminus which must be surrounded by acidic amino acids (Figure 1). Phosphorylated receptors will then bind with high affinity an uncoupling protein, called 13arrestin. [3-arrestin competes with Gs for the receptor and thereby causes uncoupling between receptors and their G-proteins. Intriguingly, ]3ARK usually occurs in the soluble fraction of cells and moves to the membrane only to phosphorylate activated receptors. This translocation is mediated by free Gprotein 137-subunits [55] and phosphatidylinositol-4,5-bisphosphate binding [56, 57]. It remains somewhat puzzling why most of the ]3ARK seems to localize to the endoplasmic reticulum when stimulated cells are investigated by immunofluorescence [58]. There are no known G-protein-coupled receptors in intracellular organelles. However, an integral membrane protein has been localized to microsomal membranes which is able to bind ]]ARK with high affinity [59]. It is also possible that ]3ARK may phosphorylate yet unknown proteins. Recently it was demonstrated that I3ARK can act as a substrate for protein kinase C [60, 61 ]. PKC-phosphorylated 13ARK will phosphorylate rhodopsin or 132-adrenergic receptor much faster, but not to a higher stoichometry. However, soluble substrates are actually phosphorylated less efficiently which suggests that phosphorylated lARK might have a higher affinity for membranes. Consistent with this finding, a soluble fusion protein which contained the C-terminus of ~ARK comprising both the phospholipid- and the G137-binding domain could
324
be phosphorylated by protein kinase C in vitro [61]. The activation of IgARK by protein kinaseC adds an additional level of complexity to short-term desensitization since desensitization is now also subject to crosstalk between various signal transduction pathways. 5. REGULATION OF G-PROTEIN ACTIVITY Heterotrimeric G-proteins do not only transduce signals from transmembrane receptors to their effectors, they also play important roles in vesicular trafficking [62]. In recent years, an increasing number of proteins have been characterized that are able to interact with G-proteins and regulate their activity [63]. It is yet unknown whether any of these proteins may interfere with signal transduction between [3-adrenergic receptors and adenylyl cyclase. However, it has been shown for one protein, phosducin, that overexpression in A431 cells affected cAMP response to 132-adrenergic agonists [64]. Phosducin is an ubiquitiously expressed protein (with the highest expression in the retina) [65, 66] which can bind G-protein 137-subunits with high affinity [67]. This interaction restricts the availability of 13y-subunits for other proteins, such as o~-subunits [68] or lARK [69]. Phosducin can be phosphorylated by protein kinase A which results in a loss of its GI3y-binding properties [65, 69]. Whether phosducin also interacts with G-protein ~-subunits is a matter of debate. Overexpression of phosducin results in complex effects on adenylyl cyclase activation by the 132-adrenergic receptor [64]. The maximal cAMP production in phosducin-overexpressing A431 cells is approximately 40% of that in wild-type cells, indicating that sequestration of GI3y subunits by phosducin blunts the signal transduction through Gs. This observation is consistent with data obtained in vitro where phosducin inhibits isoproterenol-stimulated adenylyl cyclase activity in A431 membranes [65]. However, during the first minute of stimulation, cAMP production in phosducin-overexpressing cells is increased compared to the wild type. This is due to the competition of phosducin with [3ARK for G-protein Igy subunits which results in impaired lARK activation. This is supported by the observation that desensitization is lower in membranes prepared from phosducin-overexpressing cells. Addition of PKI to permeabilized cells augmented the phosducin effects, showing that only part of the overexpressed phosducin was phosphorylated by protein kinase A. Phosducin-like protein, which was recently cloned from rat brain [70], is 65% homologous to phosducin. It is overexpressed in the brains of ethanol-treated rats [70]; the physiological significance of this observation is unclear. Recombinant phosducin-like protein does also bind to G-protein 137-subunits in vitro, although with a slightly lower affinity than phosducin [71 ]. CONCLUSION Signalling via G-protein-coupled receptors is one of the key processes regulating the activity of cells, their growth and their differentiation. This process gains an increasingly complex appearance because not only the signalling proteins themselves, but also other intracellular proteins seem to be essential components of an entire "receptor system". On the other hand, the mechanisms of signal initiation and transmission as well as those leading to signal termination and to desensitization are beginning to be unravelled at the molecular level. Undoubtedly, future research will be directed at the further elucidation of these molecular structures and mechanisms, and such data will not only help to understand one of the fundamental biological
325
processes but will also become instrumental in defining its pathology and in developing means of pharmacological interference. ACKNOWLEDGEMENTS
Figures 2 and 4 were prepared with MOLSCRIPT [72] and Raster3D [73, 74]. We thank Dr. Steven Sprang for the coordinates of the Gi heterotrimer and Stefan Danner for critical reading of the manuscript. Research in the authors' laboratory is supported by grants from the Deutsche Forschungsgemeinschaft, the Bundesministerium ftir Bildung und Forschung, the European Commission, and the Fonds der Chemischen Industrie. REFERENCES
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