Review article
Analytical pharmacology of G protein-coupled receptors by stoichiometric expression of the receptor and Gα protein subunits

https://doi.org/10.1016/S1056-8719(01)00126-5Get rights and content

Abstract

The description of a new family of recombinant proteins, which are constructed by the covalent fusion of the cDNA encoding a G protein-coupled receptor with that of a Gα protein subunit, has recently been introduced as an original strategy to explore receptor pharmacology under defined experimental conditions. As such, a controlled 1:1 stoichiometry of receptor and Gα protein expression can be achieved, as well as a forced spatial proximity to each other. Fusion proteins have been revealed as active at the receptor ligand binding level and functional at the Gα protein and effector level. Insights on analytical pharmacological data are discussed for wild-type and mutant receptors interacting with a given Gα protein subunit and different subtypes of either wild-type or mutant Gα proteins activated by a single receptor subtype. A possible alteration of the receptor:Gα protein selectivity may occur due either to the spatial proximity of both protein partners or to a constraint receptor state unable to accommodate to different Gα protein states. Coactivation of endogenous Gα proteins in host cells expressing a fusion protein has also been observed, but depends mainly on the coupling efficiency of the receptor and Gα protein engaged in the fusion process. The ligand's apparent intrinsic activity has been shown to be either enhanced, attenuated, or unmodified when the functional responses of a fusion protein are compared to the coexpression of both fusion protein partners.

Introduction

Intercellular communication may typically occur via the release of molecules by one cell, which then bind to specific receptors of an adjacent cell where they elicit a biological response. Among these receptors, the superfamily of guanine nucleotide-binding protein-coupled receptors (GPCR) is the largest and most widespread. It includes receptors for hormones, neurotransmitters, small peptides to large proteins, a number of taste and odorant molecules, ions and photons (Watson & Arkinstall, 1994). These receptors have a common structural basis: seven α-helically-oriented transmembrane domains spanning the plasma membrane, which are connected by extra and intracellular loops, a N-terminal extracellular region and a C-terminal intracellular domain. The function of GPCR is to transmit a biological signal initiated by the binding of a specific ligand to a downstream signalling pathway. They do so by interacting with a multiproteic complex: the heterotrimeric guanine nucleotide-binding protein or G protein. This complex is composed of three different subunits: α, β, and γ; each of them existing as multiple and distinct forms encoded by different genes. The combination of these three subunits can occur in multiple manners, thus, allowing the assembling of many different heterotrimeric G proteins. Some combinations of Gβ and Gγ subunits probably exist only for a short period of time due to the instability of these Gβγ dimers (Gautam, Downes, Yan, & Kisselev, 1998). The transcription pattern of each G protein subunit greatly determines its coexpression with a particular GPCR subtype (Offermans & Schultz, 1994). The biological signal resulting from the activation of a given GPCR by an agonist depends on a complex network of interactions between the receptor, Gα and Gβγ subunits of the G protein and cellular effectors. Signalling via GPCR is diverse, in particular as a single receptor subtype can activate distinct G proteins Hildebrandt, 1997, Kenakin, 1995, Pauwels, 2000. Furthermore, signalling is even more heterogeneous when considering that the amino acid sequence for one receptor subtype can vary between individuals and the increasing number of GPCR isoforms generated by alternative splicing or RNA editing of the pre-mRNA Kilpatrick et al., 1999, Pauwels, 2000.

Among several parameters that affect the output response upon GPCR stimulation by an agonist, one is the ratio between G protein and effector molecules per receptor molecule (Kenakin, 1997). The stoichiometry of the interaction between a GPCR, G protein, and effector was originally postulated to be 1:1:1 (Furchgott, 1966) and equilibrium equations were proposed in the classical ternary model (De Lean, Stradel, & Lefkowitz, 1980). This model has been accommodated to the growing knowledge on G proteins and effectors. The extended ternary complex model, proposed by Lefkowitz and colleagues Lefkowitz et al., 1993, Samama et al., 1993, is an example. It takes into account that constitutive, agonist-independent GPCR activation may exist for wild-type and mutant GPCR and that constitutive receptor activation is strongly influenced by the GPCR:G protein ratio (Kenakin, 1995). A 3- to 22-fold amplification ratio was observed between the number of cannabinoid and opioid receptors and activated G proteins in the rat brain (Sim, Selley, Xiao, & Childers, 1996). S49 lymphoma cells demonstrated a 10-fold excess in Gαs protein as compared to the endogenously expressed β2-adrenoceptor (β2 AR; Ransñas & Insel, 1988). In the neuroblastoma×glioma hybrid cell line NG108-15, the cellular level of adenylyl cyclase is presumably the limiting step in the stimulation of cAMP formation by a stably expressed β2 AR Kim et al., 1994, MacEwan et al., 1995. In this cellular system, the maximal number of Gαs protein:adenylyl cyclase complexes that could be formed was about 17,000 copies per cell, whereas the total number of Gαs protein subunits was 1 300,000 per cell (Kim et al., 1994). In expression systems where one or the other component of the signal activation cascade is more abundant, the magnitude of the observed ligand response may be strongly affected by the relative expression level of the GPCR molecule, the G protein or the effector (Kenakin, 1997). This may be worth to consider when comparing ligand responses between for instance wild-type and mutant GPCR subtypes and a respective Gα protein or when analysing the effect of mutations in a Gα protein with regard to one single GPCR subtype. One strategy to overcome stoichiometric hindrance between a receptor and a Gα protein subtype has been initially reported by Bertin, Freissmuth, Jockers, Strosberg, and Marullo (1994). These authors constructed a fusion polypeptide by covalently linking the C-terminal portion of the coding sequence of a β2 AR to the N-terminal portion of a Gαs protein subunit (Fig. 1). By this way, a fixed 1:1 stoichiometry between receptor and Gα protein was achieved. This fusion protein was analysed upon stable expression in the S49 cyc lymphoma cells, containing an endogenous β2 AR but lacking Gαs protein (cyc mutation; Bertin et al., 1994). This allowed the comparison of β2 AR and Gαs protein coexpression in wild-type S49 cells and the β2 AR:Gαs fusion protein expression in S49 cyc cells; an increased potency and maximal stimulation of adenylyl cyclase by the agonist isoproterenol was observed at the β2 AR:Gαs fusion protein (Bertin et al., 1994).

Functional responses for a number of fusion proteins have recently been obtained at the Gα protein and effector level (see Table 1); insights on the observed analytical pharmacological data will be discussed. The present review will mainly focus on the three following points: functional aspects of GPCR:Gα fusion proteins, modulation of ligand's intrinsic activity as the result of a fixed 1:1 GPCR:Gα protein stoichiometry and is a GPCR:Gα fusion protein response specific to the fused Gα protein. The reader is also referred to recent review articles on GPCR and G protein interactions Bikker et al., 1998, Gudermann et al., 2000 and on GPCR:Gα fusion proteins Milligan, 2000, Seifert, Wenzel-Seifert, & Kobilka, 1999.

Section snippets

Construction of GPCR:Gα fusion proteins

Fusion proteins can be constructed by two different molecular biology strategies. A step in common with both of them is the removal of the GPCR stop codon (see Fig. 1). In a first approach, the introduction of an enzymatic restriction site removes the stop codon by modifying it into one encoding a glycine residue for the 5-hydroxytryptamine, serotonin (5-HT1A):Gαo fusion protein (Dupuis, Tardif, Wurch, Colpaert, & Pauwels, 1999) or an alanine for the α2A AR:Gαi1, α2A AR:Gα15, and α2B AR:Gα15

Modulation of ligand's intrinsic activity as the result of a fixed 1:1 GPCR:Gα protein stoichiometry

Comparison of either ligand's potencies or maximal responses between a GPCR:Gα fusion protein and the corresponding coexpression experiment reveals three types of responses: the fusion protein response can be either enhanced, decreased, or equal to the coexpression experiment (Table 2). The β2 AR:Gαs fusion protein yielded a higher proportion (44–54%) of high affinity agonist (isoproterenol)-binding sites when stably expressed in S49 cyc and in Sf9 cells, as compared to wild-type S49 cells

Is a GPCR:Gα fusion protein response specific to the fused Gα protein?

Though the spatial structure of the GPCR portion in the fusion protein is probably not very different from a native GPCR as presumed by similar ligand binding data, conformational modifications may occur at the Gα protein moiety due to its concomitant translation following the GPCR coding portion. They can in turn influence the specificity of the interaction with a fused GPCR and the monitored downstream functional response. Moreover, interaction and subsequent activation of endogenous Gα

Conclusions

The expanding list of GPCR:Gα fusion proteins provides a new tool to examine functional responses of cell signalling systems at a strict stoichiometry of one receptor molecule for one Gα protein molecule. Though constraining the spatial structure of both the GPCR and Gα protein subunits, the fusion protein can be considered as a functional proteic entity. However, the contribution of endogenous Gα proteins in the receptor activation process of a fusion protein cannot be ruled out under certain

Acknowledgements

S. Brignatz is acknowledged for fruitful secretarial assistance.

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