Biochemical and pharmacological control of the multiplicity of coupling at G-protein-coupled receptors

Abstract

For decades, it has been generally proposed that a given receptor always interacts with a particular GTP-binding protein (G-protein) or with multiple G-proteins within one family. However, for several G-protein-coupled receptors (GPCR), it now becomes generally accepted that simultaneous functional coupling with distinct unrelated G-proteins can be observed, leading to the activation of multiple intracellular effectors with distinct efficacies and/or potencies. Multiplicity in G-protein coupling is frequently observed in artificial expression systems where high densities of receptors are obtained, raising the question of whether such complex signalling reveals artefactual promiscuous coupling or is a genuine property of GPCRs. Multiple biochemical and pharmacological evidence in favour of an intrinsic property of GPCRs were obtained in recent studies. Thus, there are now many examples showing that the coupling to multiple signalling pathways is dependent on the agonist used (agonist trafficking of receptor signals). In addition, the different couplings were demonstrated to involve distinct molecular determinants of the receptor and to show distinct desensitisation kinetics. Such multiplicity of signalling at the level of G-protein coupling leads to a further complexity in the functional response to agonist stimulation of one of the most elaborate cellular transmission systems. Indeed, the physiological relevance of such versatility in signalling associated with a single receptor requires the existence of critical mechanisms of dynamic regulation of the expression, the compartmentalisation, and the activity of the signalling partners. This review aims at summarising the different studies that support the concept of multiplicity of G-protein coupling. The physiological and pharmacological relevance of this coupling promiscuity will be discussed.

Introduction

Cell-to-cell communication is one of the principal characteristics of multicellular organisms. This process relies on the existence of a multitude of transmitters that are released from one cell and that trigger the response in others after specific recognition by specialised receptors. In addition to intracellular receptors that frequently function as transcription modulators, a large variety of cell surface receptors have been identified that can be classified in at least three different groups. The first group contains ion channels (ligand-gated ion channels) whose controlled permeability ensures fast generation of cellular responses through altered ionic fluxes. The second group are ligand-activated membrane-bound tyrosine kinases that phosphorylate key proteins frequently involved in the control of cell proliferation and differentiation. Finally, the third group contains the so-called G-protein-coupled receptors (GPCR) that trigger intracellular responses through elaborate signalling that requires the participation of GTP-binding proteins (G-proteins) and multiple membrane-bound and intracellular partners. The principal characteristic of the latter system is its complexity, which results from the intrinsic property of response amplification throughout the signalling cascade and from the diversity of regulatory mechanisms that control the nature, the amplitude, and the duration of the cellular responses.

GPCRs constitute the largest family of cell surface molecules involved in signal transmission. More than 1000 receptors for sensory (odorants, light, etc.) and chemical stimuli (catecholamines, amino acids, peptides, and even ions) have been identified that share some common structural and biochemical properties. Up to 1–5% of the total cell proteins correspond to GPCRs, and together, their genes represent up to 1% of the total genome of mammalians. Hence, they constitute one of the principal targets of drugs used in pharmacology, especially in the CNS. GPCRs are also referred to as seven-transmembrane domain receptors based on their highly conserved backbone structure (Muller, 2000). Thus, all members of this family are constituted of a single peptide containing seven hydrophobic regions of similar length separated by hydrophilic loops of variable sizes. Based on the homology with the visual pigments bacteriorhodopsin and rhodopsin, whose structures have been elucidated by crystallography Henderson et al., 1990, Okada et al., 2000, it is well established that these receptors are characterised by the presence of seven α-helices crossing the plasma membrane, separated by intracellular and extracellular loops (heptahelical receptors). The NH₂ terminus is exposed to the extracellular environment and the COOH terminus is intracellular. Despite the conservation of this original structure, sequence homologies between distinct GPCRs are generally restricted to the transmembrane domains of closely related receptor subtypes. It is generally demonstrated that the ligand recognition site involves the extracellular domains of the receptor (both the NH₂ terminus and the extracellular loops) and the pocket formed by the assembly of the seven-transmembrane helices. Interaction of the receptor with intracellular signalling partners (G-proteins) involves intracellular domains (both the COOH terminus and the intracellular loops) Gether, 2000, Wess, 1997, Wess, 1998. On the basis of structural and limited sequence similarities, mammalian GPCRs are now generally classified in at least three different groups. Group A (rhodopsin-type receptors) is the largest group and contains many receptors for classical neurotransmitters (biogenic amines and nucleotides), prostaglandins, and a large variety of peptides and neuropeptides. This first group also contains the multitude of receptors for odorants and the well-known visual pigment rhodopsin as well as the cell surface receptors for some viruses. Group B (secretin/glucagon receptors) contains the receptors for distinct hormones and peptides (e.g., calcitonin, glucagon, parathyroid hormone, secretin, vasoactive intestinal polypeptide, and diuretic hormone). Group C (frequently referred to as the metabotropic glutamate receptors group) was described more recently and contains GPCRs for the amino acids glutamate and γ-aminobutyric acid (GABA) and for the Ca²⁺ ion. For a review, see Wess (1998) as well as the GPCR database http://www.gpcr.org/7tm/ Horn et al., 1998, Horn et al., 2001.

Although emerging studies have revealed the existence of G-protein-independent signallings through some GPCRs Bockaert & Pin, 1999, Hall et al., 1999, Heuss et al., 1999, Hur & Kim, 2002, a common biochemical feature of these receptors is their interaction with G-proteins and the activation of downstream signalling cascades through a well-documented molecular mechanism. The binding of the transmitter or exogenous agonists alters the conformation of critical domains of the seven-transmembrane helix pocket, which in turn causes changes in the conformation of intracellular domains of the receptor. These changes promote the specific association of the receptor with a variety of heterotrimeric G-proteins. These are composed of an α-subunit interacting with a βγ complex. Activation of the receptor promotes the exchange of a molecule of GDP by a molecule of GTP within the active site of the α-subunit. The binding of GTP causes the dissociation of the heterotrimeric complex, and both the GTP-bound α-subunit and the released βγ complex are then able to interact with intracellular or membrane effectors (enzymes or ion channels). The intrinsic GTPase activity of the α-subunit hydrolyses GTP into GDP, restoring its initial inactive conformation as well as its affinity for the βγ complex. For many receptors, the localisation of the molecular determinants of the receptor involved in the coupling and activation of G-proteins has been investigated. With some exceptions, these studies have highlighted the role of membrane-proximal regions of the second and third intracellular loops and of the COOH terminus of the receptor in driving the coupling. However, up to now, no consensus sequences for G-protein selectivity have been clearly identified (for reviews, see Bockaert & Pin, 1999, Gether, 2000, Li et al., 1994, Wess, 1997, Wess, 1998).

The complexity and specificity of GPCR signalling partly relies on the existence of numerous closely related molecular species of the G-protein subunits Downes & Gautam, 1999, Hildebrandt, 1997, Morris & Malbon, 1999. Up to now, at least 23 α-subunits derived from 17 different genes have been identified and are classified into four families (Gα_i/o, Gα_s, Gα_q/11, and Gα₁₂) (see Table 1). Concerning β- and γ-subunits, at least 6 and 12 different molecular species have been described, respectively Gautam et al., 1998, Vanderbeld & Kelly, 2000. As summarised in Table 1, a large variety of intracellular and membrane effectors have been identified for G-proteins, and the list continues to increase (Hamm, 1998). Although all combinations do not necessarily exist in nature, the theoretical number of heterotrimeric G-protein assemblies is particularly large, and this probably contributes to the diversity and selectivity of intracellular signals activated by GPCRs. As indicated in Table 1, in comparison with the variety of receptors and G-proteins, the number of downstream effectors (enzymes and ions channels) is rather limited, and many related G-proteins (e.g., G_i-1, G_i-2, and G_i-3) ensure the coupling with the same intracellular effector (adenylate cyclase). It is also likely that a given receptor has the possibility to interact independently with many G-proteins within the same class (e.g., Albert & Robillard, 2002, Burford et al., 2000, Chalecka-Franaszek et al., 2000, Offermanns et al., 1994, Wise et al., 1999). The nature of the G-protein involved in the coupling is probably critical in modulating the efficacy and the potency of cell signalling. Within one class of G-proteins, the subtype involved in the transduction from the receptor to the effector will depend on the availability of G-proteins in the vicinity of the receptor and thus may differ from one cell to another Milligan, 1996, Neubig, 1994, Ostrom, 2002, Ostrom et al., 2000.