GPCRs comprise a wide and diverse class of eukaryotic transmembrane proteins with well-established pharmacological significance. As a consequence of recent genome projects, there is a wealth of information at the sequence level that lacks any functional annotation. These receptors, often quoted as orphan GPCRs, could potentially lead to novel drug targets. However, typical experiments that aim at elucidating their function are hampered by the lack of knowledge on their selective coupling partners at the interior of the cell, the G-proteins. Up-to-date, computational efforts to predict properties of GPCRs have been focused mainly on the ligand-binding specificity, while the aspect of coupling has been less studied. Here, we present the main motivations, drawbacks, and results from the application of bioinformatics techniques to predict the coupling specificity of GPCRs to G-proteins, and discuss the application of the most successful methods in both experimental works that focus on a single receptor and large-scale genome annotation studies.
Introduction / Background
G-protein coupled receptors (GPCRs) comprise a very important family of eukaryotic cell-surface membrane proteins. They are characterized by the structural hallmark of seven transmembrane helices, as exemplified by the crystal structure of rhodopsin (Palczewski et al. 2000), that has been extensively used as a homology modeling template for many receptor sequences (Nikiforovich et al. 2001; Becker et al. 2003). GPCRs play a pivotal role in signal transduction of eukaryotic cells, acting as the major sensors at the boundary between a cell and the outside world. Depending on their ligand-binding specificity, GPCRs can be activated by a broad range of external stimuli, from ions and small molecules to larger peptides and proteins, including light (Gether 2000). To perform these functions, GPCRs have evolved to a diversity of sequences that are traditionally classified in six major families, based mainly on shared homology (Horn et al. 2003). GPCRs have known representatives in most eukaryotic organisms, including yeast and plants, such as the recently discovered Arabidopsis thaliana seven-transmembrane (7TM) domain receptor GCR1 (Jones and Assmann 2004).
As signified by their name, upon binding to a ligand, GPCRs exert their role through the specific interaction with a more limited repertoire of intracellular proteins that hydrolyze GTP, namely the G-proteins (Neer and Clapham 1988). G-proteins are heterotrimeric complexes composed of three subunits Gα, Gβ and Gγ. They are classified into four main families, according to the type of their α-subunit, which also possesses Ras-like GTPase activity (Benjamin et al. 1995). These include Gs and Gi/o, which stimulate and inhibit adenylate cyclase, respectively (Johnston and Watts 2003), Gq/11, that activates phospholipase C (Exton 1993) and the less characterized G12/13 family that activates the Na+/H+ exchange pathway (Kurose 2003). At least 16 different subtypes of Gα subunits have been identified and classified in these four families (Downes and Gautam 1999; Kristiansen 2004). Interaction of the G-protein trimer with the activated receptor triggers the exchange of the bound GDP with GTP, and subsequently the dissociation of the complex to Gα and Gβγ moieties, that activate downstream effector molecules. Hydrolysis of GTP to GDP by the α subunit renders the complex to its original, inactive state (Neer 1995). As a result, depending on the selectivity of the GPCR - G-protein interaction, a specific downstream pathway may be activated. Despite extensive experimental and computational studies, the structural basis of this specificity is not well characterized, while the mechanisms that determine the function of the activated GPCR/G-protein complex are yet to be uncovered (Muramatsu and Suwa 2006). Furthermore, the diversity of GPCR-G-protein interactions is enriched by several receptors that may alternatively interact with more than one family of G-proteins, known as promiscuous GPCRs. For instance, the human thyrotropin receptor can couple to all four G-protein families (Laugwitz et al. 1996). In general, promiscuity seems to be a rule rather than an exception for interactions between GPCRs and G-proteins (Wess 1998; Oliveira et al. 1999; Horn et al. 2000). Several lines of evidence indicate the importance of the GPCR intracellular regions, as well as the intracellular boundaries of the transmembrane helices (Gether 2000). It is also established that the regions of interaction on the G-protein are mainly the N-terminus of the Gα and the N- and C-termini of Gγ subunit. However, up to date, these findings have not been incorporated to a high-resolution, systematic model of GPCR – G-protein interactions, while the nature of the underlying mechanism is believed to be specific to the interacting partners (Wess 1998).
Key Terms in this Chapter
G-Protein Coupled Receptors (GPCRs): Also known as seven transmembrane (heptahelical) receptors, due to their characteristic membrane topology (seven transmembrane helices, extracellular N-terminus and intracellular C-terminus). They are transmembrane proteins acting as the sensory component of cellular signalling pathways. GPCRs, are a key class of eukaryotic membrane receptors and roughly 50% of all small molecule therapeutics target GPCRs. Vision, smell and some of taste uses GPCRs. Ligands for GPCRs cover a wide range of organic chemical space, including proteins, peptides, sugars, amines and amino-acids, nucleotides, lipids and more. They transduce signals from extracellular space into the cell, through their interaction with G proteins, which act as switches forming hetero-trimers composed of different subunits (a,ß,?). Two GPCRs’ crystal structures are currently available, the structure of Rhodopsin and the recently solved three-dimensional structure of beta-2 Adrenergic Receptor.
Hidden Markov Models (used herein): Probabilistic models widely used for describing features of a protein sequence. Hidden Markov Models introduce a “regular grammar” that characterizes a set of biological sequences. These are generative models, which renders them highly applicable in biological sequence analysis. In general, a HMM is composed of a set of states that form a first order Markovian process, connected by means of the transition probabilities. Each state, has a unique probability distribution for generating (emitting) the symbols of the finite alphabet (nucleotides or amino-acids). The most widely used variant of Hidden Markov Model (HMM) is the profile HMM which models in a probabilistic manner the matches, inserts and deletions occurring in every column of a multiple sequence alignment. However, other variations are also common (i.e. the circular HMM).
Orphan Receptors: GPCRs for which no information on their ligand or coupling specificity is available. These are usually identified as a result of genome sequencing projects and large efforts are undertaken to functionally characterize them.
Coupling Selectivity: G protein trimers are named after their a-subunits, which on the basis of their amino acid similarity and, most importantly by their cellular function, are grouped into four families. These include, Gas and Gai/o, which stimulate and inhibit respectively adenylate cyclase, Gaq/11 which stimulates phospholipase C, and the less characterized Ga12/13 family that activates the Na+/H+ exchanger pathway. The specificity of the interaction of a given GPCR with the pool of available intracellular G-proteins is termed coupling selectivity or specificity. The ability of certain GPCRs to interact with more that one types of G-proteins (i.e. Gas and Gai/o) is known as promiscuous coupling selectivity. GPCRs coupled to members of the Ga12/13 family are all exhibiting promiscuous coupling preferences.
Genome Annotation: The functional characterization (by means of biochemical experiments or computational prediction algorithms) of novel genes in newly sequenced and assembled genomes.
G-Proteins: The term is used to describe GTP-binding proteins. There are two classes of G-proteins, the small cytoplasmic G-proteins (Gh) and the hetero-trimeric G-proteins composed of different subunits (a,ß,?) that mediate the signal of heptahelical receptors (GPCRs). Agonist binding to GPCRs leads to association of the hetero-trimeric G protein with the receptor, GDP-GTP exchange in the G protein a subunit followed by dissociation of the G protein into a-GTP and ß? complexes. The dissociated subunits can activate or inhibit several effectors such as adenylyl cyclase, PLCß, tyrosine kinases, phosphodiesterases, phosphoinositide 3-kinase, GPCR kinases, ion channels, and molecules of the mitogen-activated protein kinase pathway, resulting in a variety of cellular functions. However, there is evidence that some GPCRs transduce their signal through in a way that is not G protein-dependent, and also that hetero-trimeric G proteins are involved in mediating the action of single-spanning membrane receptors.