Fusion proteins between GPCRs and G proteins

Wild-type and mutant GPCRs may show constitutive activity, and the extended ternary complex model (Samama et al., 1993) reveals that this activity is influenced by the GPCR:G protein ratio. As shown above, the GPCR:G protein ratio may also influence the cellular response to a given agonist. Yet, GPCR and Ga protein densities may differ considerably from one cell type to another and (especially in the case of transient transfections) even from one day to another. In theory, this may lead to quite some variation in the agonist dose-response curves and in the receptor basal activities when comparing different experiments.

Start Mutated stop Slop codon codon codon

Start Mutated stop Slop codon codon codon

Figure 184 Schematic presentation of a GPCR-Ga fusion protein. Reprinted from Journal of Pharmacology and Toxicological Methods, 45, Wurch, T. and Pauwels, P. J., Analytical pharmacology of G protein-coupled receptors by stoichiometric expression of the receptor and G(alpha) protein subunits, 3-16. Copyright (2001), with permission from Elsevier.

Figure 184 Schematic presentation of a GPCR-Ga fusion protein. Reprinted from Journal of Pharmacology and Toxicological Methods, 45, Wurch, T. and Pauwels, P. J., Analytical pharmacology of G protein-coupled receptors by stoichiometric expression of the receptor and G(alpha) protein subunits, 3-16. Copyright (2001), with permission from Elsevier.

One strategy to overcome this potential cause of variability is to create fusion proteins by covalently linking the C-terminal portion of a GPCR to the N-terminal portion of a Ga protein subunit (Figure 184). By this way, a fixed 1:1 stoichiometry between receptor and Ga is achieved. Although this stoichiometry might not be the same as in physiological systems, it remains the same irrespective of the cell system in which the fusion protein is expressed and of the absolute level of expression.

According to its proponents, the major benefit of the GPCR-Ga fusion protein approach is that it should allow an accurate comparison of experimental data obtained in different laboratories. This is obviously quite a difficult task when dealing with traditional cellular systems in which receptors and G proteins are expressed individually. In favour of this claim, it has been shown that the a2A-adrenergic receptor activation profile by a series of full and partial agonists was not affected over a 30-fold range of expression of the receptor-Ga15 fusion protein, whereas an enhancement of the maximal response of partial agonists was observed when the free receptor was co-expressed with increasing amounts of Ga15 (Figure 185).

The 1:1 stoichiometry should even allow the comparison of agonist intrinsic efficacies, provided that the measured response is measured at the point of GPCR-G protein interaction (e.g. by [35S]GTPyS binding) rather than at some downstream point. Under these conditions, the intrinsic activity of a ligand (a) should not yet be corrupted by non-linear stimulus-effect coupling (i.e. 'receptor reserve') so that it should reflect the intrinsic efficacy (e) of that ligand.

A major premise of the GPCR-Ga fusion protein approach is that fusion promotes efficient coupling without altering the fundamental properties of the signalling partners. From a structural point of view, several native Ga proteins have been found to bear a myristyl or palmitoyl fatty acid side chain. This post-translational modification allows the Ga protein to be attached to the plasma membrane and, hence, to reside in the

Figure 185 Intrinsic activity of a2-adrenergic receptor Ligands in transfected CHO cells as a function of (Left) the receptor-Go^ fusion protein concentration or (right) Ga15 protein plus a fixed concentration of receptor. Reprinted from Journal of Pharmacology and Toxicological Methods, 45, Wurch, T. and Pauwels, P. J., Analytical pharmacology of G protein-coupled receptors by stoichiometric expression of the receptor and G(alpha) protein subunits, 3-16. Copyright (2001), with permission from Elsevier.

Figure 185 Intrinsic activity of a2-adrenergic receptor Ligands in transfected CHO cells as a function of (Left) the receptor-Go^ fusion protein concentration or (right) Ga15 protein plus a fixed concentration of receptor. Reprinted from Journal of Pharmacology and Toxicological Methods, 45, Wurch, T. and Pauwels, P. J., Analytical pharmacology of G protein-coupled receptors by stoichiometric expression of the receptor and G(alpha) protein subunits, 3-16. Copyright (2001), with permission from Elsevier.

proximity of GPCRs. In the same vein, fusion to a GPCR also produces a membrane anchor for Ga and, in theory, fusion proteins can increase the efficiency of Ga activation by a GPCR because they are even closer to each other, as in the case of the individual proteins. In support of his assertion, fusion has been shown to rescue functional interactions of a myristoylation-deficient Gau mutant with the 02A-adrenergic receptor.

In practice, however, there are several points of concern with regard to this approach:

• GPCR-Ga fusion proteins often generate signals that resemble those of the free receptors. However, upon close comparison of ligand potencies or maximal responses between a fusion protein and the corresponding co-expression system, it turns out that the fusion protein response can be either enhanced, decreased or equal to the co-expression experiment (Table 22).

• The Ga-Py interaction may be attenuated by the fusion process. Whereas Py contributes to the interaction of Ga with the receptor under normal circumstances, the effects of Py on the responsiveness of a fusion protein are usually of low magnitude. In this respect, GPCRs interacting with Ga; are known to stimulate the MAPK signalling pathway upon agonist activation via release of Py subunits. The fusion constructs could be unable to signal down the ERK-MAPK cascade because the Gai protein is tethered to the receptor and is therefore no longer able to interact with endogenous Py subunits.

• Special care should be taken with PTX-resistant GPCR-GajCys3S1Gly fusion proteins since the mutation could produce a suboptimal GPCR-Ga interface. Whereas full agonists can produce sufficient conformational alterations in a GPCR to overcome this handicap, partial agonists might be less effective in doing so. This might result in a lower efficacy of partial agonists (Figure 186).

Table 22 Comparison between R-Ga fusion proteins and co-expressed R and Ga. Reprinted from Journal of Pharmacology and Toxicological Methods, 45, Wurch, T. and Pauwels, P. J., Analytical pharmacology of G protein-coupled receptors by stoichiometric expression of the receptor and G(alpha) protein subunits, 3-16. Copyright (2001), with permission from Elsevier.

Table 22 Comparison between R-Ga fusion proteins and co-expressed R and Ga. Reprinted from Journal of Pharmacology and Toxicological Methods, 45, Wurch, T. and Pauwels, P. J., Analytical pharmacology of G protein-coupled receptors by stoichiometric expression of the receptor and G(alpha) protein subunits, 3-16. Copyright (2001), with permission from Elsevier.

GPCR and Ga Evaluation test

Ligand

EC50

Maximal

protein combination

response

Fusion protein > coexpression

ß2 AR and GasS cAMP formation

isoproterenol

Fusion 45 nM

100%

ß2 AR + Gas;

58%

650 nM

Coexpression > fusion protein

a2A AR and Ga15 Ca2+ mobilisaiion

clonidine

Fusion 58 nM

100%

®2A AR + Gai5;

170%

6.0 nM

Fusion protein = coexpression

5-HT1A and GTPase

5-HT

Fusion 100 nM

100%

GaiiCys351Gly

5-HT1A; 80 nM

100%

GPCRs that are fused via their C-terminus to other proteins, such as green fluorescent protein, still appear to interact with and activate cellular G proteins. In the same way, agonist occupancy of a GPCR-Ga fusion protein can still cause the activation of endogenous G proteins. Hence, due attention must be given to which

Figure 186 Capacity of different a2A-adrenergic agonists to stimulate the GTPase activity of endogenous Gj-proteins and a2A receptor-GajCys351Gly fusion proteins in RAGI 77 cell membranes (Burt et al., 1998, reproduced by permission of the American Society for Biochemistry and Molecular Biology).

G protein is responsible for the observed activity. In this respect, it has also been reported that the Ga selectivity of fusion proteins could be different as compared to the free GPCR. For example, whereas isolated a2A-adrenergic receptors were shown to activate endogenous Gs (i.e. following pertussis toxin treatment, adrenaline stimulated the adenylate cyclase activity), the receptor-GajCys351Gly fusion proteins were unable to do so. Several strategies have been adopted to limit the activation of endogenous Ga of the host cells; these include:

- Careful selection of the model system. Based on the observation that mammalian GPCRs couple poorly to endogenous insect Ga proteins, Sf9 insect cells are routinely used as a host for mammalian GPCRs and GPCR-Ga fusion proteins. In the same spirit, S49 cyc~ mouse lymphoma T cells do not express functional Gas. Therefore, they constitute a host of choice for GPCR-Gas fusion proteins.

- Transfection with GPCR-Ga fusion proteins, which are naturally resistant to PTX, and treatment of the host cells with PTX to eliminate any potential interactions with endogenously expressed Gai. In this respect, certain Ga proteins (Ga15, Ga16) are naturally resistant to PTX since they do not possess the required ADP-ribosylation site. Alternatively, Ga; proteins may be mutated to become PTX-resistant (e.g. GaiCys351Gly).

Contrary to the belief that short-range interactions between GPRC and Ga may favour constitutive receptor activation, this phenomenon is not necessarily observed with GPCR-Ga fusion proteins. If observed, the constitutive activity of a receptor is likely to depend on the nature of the Ga involved. For example (Figure 187):

Figure 187 Effect of isoproterenol (ISO, ß-adrenergic agonist) and ICI 118,551 (inverse agonist) on [35S]GTPyS binding to Sf9 cell membranes bearing ß2-adrenergic receptor-Gas fusion proteins. GasL is the long form of Gas while GasS is the short form. Reproduced from WenzelSeifert, K. and Seifert, R. (2000) Molecular Pharmacology, 58, 954-966, with permission from the American Society for Pharmacology and Experimental Therapeutics.

Figure 187 Effect of isoproterenol (ISO, ß-adrenergic agonist) and ICI 118,551 (inverse agonist) on [35S]GTPyS binding to Sf9 cell membranes bearing ß2-adrenergic receptor-Gas fusion proteins. GasL is the long form of Gas while GasS is the short form. Reproduced from WenzelSeifert, K. and Seifert, R. (2000) Molecular Pharmacology, 58, 954-966, with permission from the American Society for Pharmacology and Experimental Therapeutics.

• When expressed in insect Sf9 cells, p2-adrenergic receptor-GasL fusion proteins have been shown to display constitutive activity: [35S]GTPyS binding to the G protein took place in the absence of agonist and this event was inhibited by the inverse agonist ICI 118,551.

• When expressed in the same cells, p2-adrenergic receptor-GasS fusion proteins were devoid of constitutive activity since ICI 118,551 did not affect [35S]GTPyS binding under basal conditions.

Because of the inherent 1:1 stoichiometry of GPCR-Ga fusion proteins, one could expect them to display a single high-affinity state for agonist binding. Yet this is not the case in practice. GPCR-G protein fusion proteins maintain both high and low agonist affinity states and as such behave like the co-expressed individual proteins. In this respect, it was shown that the p2-adrenergic receptor couples much more efficiently to the Gas-fusion partner than to Ga; and Gaq partners in insect Sf9 membranes (Figure 188). Although further experimental confirmation may be necessary, these findings suggest

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-11 -10-9 -e -7 -6 -5 -4-11-10-9 -8 -7 -6 -5 -4 Isoproterenol (Loq M)

Figure 188 Isoproterenol competition binding curves with insect Sf9 membranes expressing P2 receptor-GasL and -GasS fusion proteins are biphasic. GTPyS converts the receptors into a single population with low agonist affinity. The agonist displays only low affinity in membranes expressing p2 receptor-Gai2 and -Gaq fusion proteins. Reproduced from Wenzel-Seifert, K. and Seifert, R. (2000) Molecular Pharmacology, 58, 954-966, with permission from the American Society for Pharmacology and Experimental Therapeutics.

that membrane compartmentalization and stoichiometric limitation in the amount of available Ga proteins (Section 4.10) do not constitute the sole explanations for the presence on membranes of receptor sites with high and low agonist affinities.

4.13 Multiple receptor conformations

It is becoming increasingly clear that the two-state model cannot sufficiently explain the complex behaviour of GPCRs. In physicochemical terms, molecules are considered to exist in a very large number of conformational states, and trying to describe the properties of a receptor with just two states must be an approximation. Different receptor states may be regarded to constitute minima of an 'energy landscape'. At rest, most of the receptors should be in the 'inactive' state. A spectrum of 'active' states is supposed to exist and, upon ligand binding, the population of some of these 'active' states might increase in a ligand-specific manner (Figure 189).

The ability of receptors to dimerize, to internalize, to be phosphorylated, to be desensitized and to interact with other membrane proteins can sometimes be dissociated from the activation of G proteins. For example (Figure 190), some Tyr4- and Phe8-substituted Ang II analogues promote AT1a receptor internalization (confocal microscopy of enhanced green fluorescent protein-tagged receptor) without phospholipase C signalling in CHO cells. It is difficult to explain within a simple

Figure 189 Relative abundance of 'inactive' (Ri) and 'active' (Ra) receptor states at rest and in the presence of two different agonists. Reproduced from Kenakin, T. (1996) Pharmacological Reviews, 48, 413-463, with permission from the American Society for Pharmacology and Experimental Therapeutics.

Figure 190 Comparison of ATj receptor activity and internalization in the presence of different Sar1-Ang II analogues (denomination: e.g. G48 = substitution with glycine at positions 4 and 8, A = alanine, I = isoleucine). Reproduced from Holloway, A. C., Qian, H., Pipolo, L., Ziogas, J., Miura, S.-I., Karnik, S., Southwell, B. R., Lew, M. J. and Thomas, W. G. (2002) Molecular Pharmacology, 61, 768-777, with permission from the American Society for Pharmacology and Experimental Therapeutics.

Figure 190 Comparison of ATj receptor activity and internalization in the presence of different Sar1-Ang II analogues (denomination: e.g. G48 = substitution with glycine at positions 4 and 8, A = alanine, I = isoleucine). Reproduced from Holloway, A. C., Qian, H., Pipolo, L., Ziogas, J., Miura, S.-I., Karnik, S., Southwell, B. R., Lew, M. J. and Thomas, W. G. (2002) Molecular Pharmacology, 61, 768-777, with permission from the American Society for Pharmacology and Experimental Therapeutics.

two-state model that these events do not coincide. To accommodate these findings, it might be necessary to consider that:

• Each function of the receptor is triggered by a broadly defined continuum of conformations instead of only one well-defined conformation.

• Conformations allowing GPCR phosphorylation, internalization and desensitiza-tion processes only partly overlap those activating G protein.

• Each ligand might only stabilize a certain subset of conformations.

Multistate models in which distinct conformations of the receptor are involved in coupling with distinct G proteins have been proposed. These models arose, in the first place, from the failure of the 'classical' two-state models to explain the reversals of ligand potency and/or intrinsic efficacy orders that can sometimes be observed when comparing two types of responses (involving distinct types of G proteins) that are generated by a single receptor. Some of the representative experiments are presented below:

• Human 5-HT2C receptors expressed in CHO cells were found to trigger inositol phosphate accumulation and phospholipase A2-mediated arachidonic acid release (Holloway et al., 2002). Different agonists displayed the same potency for triggering both responses, but their relative efficacies differed depending on the response (Figure 191). Some agonists (e.g., 3-trifluoromethylphenyl-pipera-zine) preferentially activated inositol phosphate accumulation, whereas others (e.g. LSD) favoured arachidonic acid release. These data reflect true differences at the level of the agonist-receptor interaction if the stimulus-response relationship is linear for both signal transduction pathways. This was ascertained by experiments where some of the receptors were irreversibly inactivated with

Figure 191 Effect of different agonists on the inositol phosphate accumulation in, and arachi-donic acid release by, human 5-HT2C receptor-expressing CHO cells. Reproduced from Berg, K. A., Maayani, S., Goldfarb, J., Scaramellini, C., Leff, P. and Clarke, W. P. (1998) Molecular Pharmacology, 54, 94-104, with permission from the American Society for Pharmacology and Experimental Therapeutics.

Figure 191 Effect of different agonists on the inositol phosphate accumulation in, and arachi-donic acid release by, human 5-HT2C receptor-expressing CHO cells. Reproduced from Berg, K. A., Maayani, S., Goldfarb, J., Scaramellini, C., Leff, P. and Clarke, W. P. (1998) Molecular Pharmacology, 54, 94-104, with permission from the American Society for Pharmacology and Experimental Therapeutics.

the covalently binding drug, phenoxybenzamine (Figure 192). Under the same experimental conditions, this treatment produced a similar reduction in the maximal 5-HT-induced inositol phosphate accumulation and arachidonic acid release without affecting the EC50 of 5-HT for either pathway.

• Transfected PACAP receptors were found to trigger Gs-mediated cAMP accumulation and Gq/11-mediated inositol phosphate accumulation. Whereas PACAP1-27 was more potent than its analogue PACAP1-38 with regard to their ability to stimulate

Figure 192 Irreversible inactivation of some of the 5-HT2C receptors in CHO cells with phenoxybenzamine (PBZ) decreases the 5-HT-mediated inositol phosphate accumulation and arachidonic acid release to the same extent. Reproduced from Berg, K. A., Maayani, S., Goldfarb, J., Scaramellini, C., Leff, P. and Clarke, W. P. (1998) Molecular Pharmacology, 54, 94-104, with permission from the American Society for Pharmacology and Experimental Therapeutics.

Figure 192 Irreversible inactivation of some of the 5-HT2C receptors in CHO cells with phenoxybenzamine (PBZ) decreases the 5-HT-mediated inositol phosphate accumulation and arachidonic acid release to the same extent. Reproduced from Berg, K. A., Maayani, S., Goldfarb, J., Scaramellini, C., Leff, P. and Clarke, W. P. (1998) Molecular Pharmacology, 54, 94-104, with permission from the American Society for Pharmacology and Experimental Therapeutics.

Figure 193 Effect of the agonists PACAP27 and PACAP38 on inositol phophate and cAMP accumulation in pituitary adenylate cyclase-activating polypeptide (PACAP) receptor-expressing LLC PK1 cells. Reprinted by permission from Macmillan Publishers Ltd: Nature, 365, Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P. H. and Journot, L., Differential signal transduction by five splice variants of the PACAP receptor, 170-175, © (1993).

Figure 193 Effect of the agonists PACAP27 and PACAP38 on inositol phophate and cAMP accumulation in pituitary adenylate cyclase-activating polypeptide (PACAP) receptor-expressing LLC PK1 cells. Reprinted by permission from Macmillan Publishers Ltd: Nature, 365, Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P. H. and Journot, L., Differential signal transduction by five splice variants of the PACAP receptor, 170-175, © (1993).

cAMP accumulation, the potency order of these agonists was reversed when measuring their ability to trigger inositol phosphate accumulation (Figure 193).

• 'Antagonists' have also been found to display distinct potencies for blocking the diverse signals that may elicited by the same agonist. Such behaviour has been observed for, for example, the CCK2 receptor in CHO cells (Figure 194). In these experiments, inositol phosphate formation and arachidonic acid release in response to the same agonist were inhibited with the same potency by L365260, but with different potencies by RB213.

Figure 194 Effect of specific CCKB receptor antagonists on CCK8-mediated inositol phosphate accumulation and arachidonic acid release. Reprinted from Journal of Neurochemistry, 73, Pommier, B., Da Nascimento, S., Dumont, S., Bellier, B., Million, E., Garbay, C., Roques, B. P. and Noble, F., The cholecystokinin B receptor is coupled to two effector pathways through pertussis toxin-sensitive and -insensitive G proteins, 281-288. Copyright (1999) Blackwell Publishing.

Figure 194 Effect of specific CCKB receptor antagonists on CCK8-mediated inositol phosphate accumulation and arachidonic acid release. Reprinted from Journal of Neurochemistry, 73, Pommier, B., Da Nascimento, S., Dumont, S., Bellier, B., Million, E., Garbay, C., Roques, B. P. and Noble, F., The cholecystokinin B receptor is coupled to two effector pathways through pertussis toxin-sensitive and -insensitive G proteins, 281-288. Copyright (1999) Blackwell Publishing.

Table 23 Receptors with alluded ligand-selective conformations. Reprinted from Trends in Pharmacological Sciences, 24, Kenakin, T., Ligand-selective receptor conformations revisited: the promise and the problem, 346-354. Copyright (2003), with permission from Elsevier.

Agonist-induced stimulus trafficking

Agonist-selective antagonist potency

PACAP receptor

CCK2 receptor

Dopamine D2 receptor

5-HT1A receptor

Drosophila tyramine receptor

ßj-Adrenoceptor

NK1 receptor P2-Adrenoceptor

Muscarinic acetylcholine receptor Calcitonin receptor Adenosine A1 receptor

Receptor internalization

CCK receptor Opioid peptide receptor Angiotensin II receptor Chemokine CCR5

a2A-Adreno ceptor Bombesin receptor Parathyroid hormone receptor

PTH-1 receptor Opioid peptide receptor

5-HT2A receptor

Receptor phosphorylation and desensitization

Cannabinoid receptor

Angiotensin II receptor

Chemokine CXCR2

Opioid peptide receptor

Delta opioid peptide receptor

5-HT3 receptor

To explain such findings, it has been assumed that agonists may trigger/stabilize specific receptor conformations (Table 23) and, in this way, modulate the receptor's preference for certain G proteins. The initial models by Kenakin (1995b) and ensuing models are largely based on the following premises:

• Different active conformations of the receptor do exist.

• Each agonist should promote its own specific active receptor conformation, a phenomenon that is commonly referred to as 'signalling-selective agonism', 'biased agonism', 'agonist-specific trafficking of receptor signalling' or simply

'agonist trafficking'.

• Each active conformation has its own G protein preference. This notion is supported by experiments with mutated p2-adrenergic receptors, which suggest that distinct intracellular receptor domains interact with each type of G protein.

'Agonist trafficking' implies that pharmacological diversity may be achieved through a single receptor by compounds that trigger distinct effector pathways (Figure 195). Obviously, this may have major positive consequences for the development of signalling-specific therapeutics (Figure 196). On the negative side, predictions of the clinical efficacy of drug constituents become more difficult when simply based on assays with recombinant cell systems. Moreover, whereas receptor subtypes might show up as single 'species' when investigating their antagonist binding properties, they might

Figure 195 Agonist trafficking. Situation for two active receptor conformations, each with a specific G protein signalling. Top: non-selective agonists stabilize both active conformations equally well. Bottom: signalling selective agonists preferentially stabilize one of the active conformations. Reprinted from Pharmacology and Therapeutics, 99, Hermans, E., Biochemical and pharmacological control of the multiplicity of coupling at G protein-coupled receptors, 25-44. © (2003), with permission from Elsevier.

Figure 195 Agonist trafficking. Situation for two active receptor conformations, each with a specific G protein signalling. Top: non-selective agonists stabilize both active conformations equally well. Bottom: signalling selective agonists preferentially stabilize one of the active conformations. Reprinted from Pharmacology and Therapeutics, 99, Hermans, E., Biochemical and pharmacological control of the multiplicity of coupling at G protein-coupled receptors, 25-44. © (2003), with permission from Elsevier.

y— Active state A Endogenous Subtype- Active state-t—-Subtype 1-—^—■ Active state B agonist selective selective / agonist agonist j--Subtype 2—Active state A

V-Subtype 3---- Active state A

\ Active state B

\-Subtype 4—Active state A

^-Active state B

Figure 196 Ligand-selective receptor conformations: drugs could be designed to modify specific physiological effects of a given receptor. Reprinted from Trends in Pharmacological Science, 24, T. Kenakin, Ligand-selective receptor conformations revisited: the promise and the problem, 346-354. Copyright (2003), with permission from Elsevier.

be split into many more pharmacologically distinct 'species' when investigating their interaction with agonists (Figure 197).

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