Trends in Pharmacological Sciences
ReviewImportance of the extracellular loops in G protein-coupled receptors for ligand recognition and receptor activation
Introduction
G protein-coupled receptors (GPCRs) form a large family of transmembrane (TM) proteins that convey an extracellular signal exerted by a hormone or neurotransmitter to an intracellular response through G proteins. They all have a similar structure, with an extracellular N-terminus, 7 transmembrane helices connected by three extracellular (EL1–3) and three intracellular loops, and an intracellular C-terminus. The GPCR super-family consists of five main classes, of which the class A (or rhodopsin-like) GPCRs form by far the largest subfamily [1].
Next to the N- and C-termini, the extracellular loops of GPCRs are the most variable structural elements of the receptor, differing greatly in length and sequence. Even within subfamilies, the extracellular loops often show low sequence homology (if any at all). Also, early data on receptor architecture stemming from bacteriorhodopsin and bovine rhodopsin provided limited and incomplete information regarding these more flexible GPCR domains. This paucity of data and ambiguity meant that structural studies of the function and activation (through, for example, mutagenesis) of receptors focused on the more conserved and better characterized regions of the receptor (e.g. transmembrane domains) [2]. As a consequence, the average ‘textbook model’ states that mainly two domains are determinants for receptor activation. That is, the region in which the ligand binds and the domain that interacts with the G protein. In this view, the extracellular loops are mainly regarded as peptide linkers to hold the functionally important transmembrane helices together and keep these stably positioned in the cell membrane. However, over the last decade, it has become clear that the extracellular loops fulfil important functional roles in receptor activation and in ligand binding. For example, several somatic mutations in the loops have been linked to disease 3, 4, 5. Therefore, the purpose of this review is to provide evidence that these neglected receptor domains are vital for the appropriate recognition and function of receptors.
Section snippets
Extracellular loops as seen in crystal structures
GPCR crystallization is extremely challenging because GPCRs are: (i) unstable outside the cell membrane, and (ii) known to adopt many conformational states. The relatively unstructured loops add to the conformational diversity. This combination of fragility and flexibility is a major hurdle in obtaining good-quality crystals. Nevertheless, we now have access to a handful of GPCR structures [6]. From these structures it is apparent that the extracellular regions can adopt very different
The first extracellular loop (EL1) provides structure to the extracellular complex
The first extracellular loop of class-A GPCRs is usually very small, consisting of only a few amino acids. As in all three loops, its amino-acid sequence is highly variable among family members. However, the length of the first extracellular loop is highly conserved (Figure 3), with >70% of class-A GPCRs having 52 amino acids separating the two most conserved residues in helices 2 and 3 (2.50 and 3.50) according to the Ballesteros and Weinstein notation [16]. Similar analyses on other GPCR
An all-encompassing role for the second extracellular loop (EL2)
The second extracellular loop in class-A GPCRs is the largest and most divergent of the three. It can differ greatly (even within subfamilies) in length and in sequence (Figure 3). In class B and class C GPCRs, this difference in length is much less pronounced. These families are much smaller than class A GPCRs, in particular family C, which consists of only 23 human receptors. In contrast, the receptors in class-B and class-C GPCRs all have large N-termini that mainly determine ligand
The third extracellular loop (EL3) influences the binding and activation of ligands
The third extracellular loop (EL3) is perhaps the least investigated of the three loops. Like EL1, it is small in all class-A GPCRs (Figure 3). This is also the case in class-B and class-C GPCRs. Nonetheless, the third extracellular loop has been proposed to be important in GPCR signalling 19, 26, 52. Claus et al. identified the presence of a hydrophobic cluster of amino-acid residues within EL3 of the thyrotropin receptor that strongly influences signal transduction and activation of G
Antibodies against the extracellular loops
Many commercially available monoclonal antibodies for GPCRs are raised against epitopes located at the extracellular surface of the receptor, most often EL2. However, these antibodies are frequently receptor conformation-dependent and have met with limited success, for example, in immuno-histochemistry [56]. Owing to subtle changes in receptor conformation, segments of the loop become more or less accessible. If the flexible EL2 moves to interact with the ligand, then the necessary epitopes for
Concluding remarks
All three extracellular loops are relevant for the activation mechanism of class-A GPCRs. EL1 probably provides structure to the extracellular region of the ligand binding site and enables movement of the transmembrane helices upon ligand binding. EL2 seems to play the most important part in activation because it is involved in direct binding of the ligand, ligand recognition, and ligand entry. It might also host allosteric binding sites. EL3 seems to be essential for appropriate folding and
Acknowledgements
We are grateful for the financial support provided by the Dutch Top Institute Pharma (project D1-105). We are very thankful to Professor Stevens from the Department of Molecular Biology at the Scripps Research Institute (San Diego, CA, USA) for providing us with the coordinates of the CXCR4 crystal structure ahead of publication. We thank Bas Vroling and the GPCRDB (http://www.gpcr.org/7tm/) for help with the bioinformatics, and Veli-Pekka Jaakola for providing Figure 4.
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