GPCRs exhibit a common architecture of seven transmembrane helices (TMs) linked by intracellular loops and extracellular loops (ECLs). Given their peripheral location to the site of G-protein interaction, it might be assumed that ECL segments merely link the important TMs within the helical bundle of the receptor. However, compelling evidence has emerged in recent years revealing a critical role for ECLs in many fundamental aspects of GPCR function, which supported by recent GPCR crystal structures has provided mechanistic insights. This review will present current understanding of the key roles of ECLs in ligand binding, activation and regulation of both family A and family B GPCRs. LINKED ARTICLESThis article is part of a themed section on the Molecular Pharmacology of G Protein-Coupled Receptors (GPCRs). To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-6. To view the 2010 themed section on the same topic visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2010.159.issue-5/issuetoc Abbreviations A2AR, A2A adenosine receptor; AT1R, angiotensin II type 1 receptor; b1AR, b1-adrenergic receptor; b2AR, b2-adrenergic receptor; C5aR, complement factor 5a receptor; CAM, constitutively activating mutation; CGRP, calcitonin gene-related peptide; CLR, calcitonin receptor-like receptor; CRF, corticotropin-releasing factor; D3R (D2R), D3 (D2) dopamine receptor; GLP-1, glucagon-like peptide-1; ECL, extracellular loop; H1R, histamine H1 receptor; HIV-1, human immunodeficiency virus type 1; M2R (M4R), M2 (M4) muscarinic acetylcholine receptor; NDI, nephrogenic diabetes insipidus; PACAP, pituitary adenylyl cyclase-activating peptide; PTH, parathyroid hormone; TM, transmembrane helix; V1aR, V1a vasopressin receptor; V2R, V2 vasopressin receptor IntroductionGPCRs form the largest class of membrane proteins in the human genome, with >800 unique receptors. They are central to cell signalling and are of great commercial value to the pharmaceutical industry worldwide, with~50% of clinically marketed drugs and~25% of top-selling drugs targeting this receptor family (Lagerström and Schiöth, 2008). GPCRs are activated by a wide variety of agonists which differ with respect to chemical class, physical properties and size -from photons and small biogenic amines to peptides and large glycoproteins (Hill, 2006).Historically, it was envisaged that binding any agonist induced the 'on' conformation that activated a single G-protein type to initiate an intracellular signal. It is now recognized that GPCR signalling is much more complex than this. Individual GPCRs can activate multiple types of G-protein, not just one type, and signalling can be G-protein independent, such as b-arrestin-dependent GPCR activation of MAPK (Azzi et al., 2003). Furthermore, there is compelling evidence to indicate that the classification of an individual ligand can be dictated by the signalling system being observed. For example, the peptide ligand SP-G is an antagonist for V 1a vasopressin receptor (V1aR) inositol phos...

G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins and are an important target for therapeutic drugs. These receptors are highly dynamic proteins sampling a range of conformational states in order to fulfil their complex signalling roles. In order to fully understand GPCR signalling mechanisms it is necessary to extract the receptor protein out of the plasma membrane. Historically this has universally required detergents which inadvertently strip away the annulus of lipid in close association with the receptor and disrupt lateral pressure exerted by the bilayer. Detergent-solubilized GPCRs are very unstable which presents a serious hurdle to characterization by biophysical methods. A range of strategies have been developed to ameliorate the detrimental effect of removing the receptor from the membrane including amphipols and reconstitution into nanodics stabilized by membrane scaffolding proteins (MSPs) but they all require exposure to detergent. Poly(styrene-co-maleic acid) (SMA) incorporates into membranes and spontaneously forms nanoscale poly(styrene-co-maleic acid) lipid particles (SMALPs), effectively acting like a 'molecular pastry cutter' to 'solubilize' GPCRs in the complete absence of detergent at any stage and with preservation of the native annular lipid throughout the process. GPCR-SMALPs have similar pharmacological properties to membrane-bound receptor, exhibit enhanced stability compared with detergent-solubilized receptors and being non-proteinaceous in nature, are fully compatible with downstream biophysical analysis of the encapsulated GPCR.

A novel DNA sensing method based on LD spectroscopy and using bionanoparticle scaffolds is described, as demonstrated by the rapid detection of DNA strands associated with bacterial and viral pathogens.

Using the GPCR rhodopsin as an exemplar, SMA SMI and DIBMA constitute a ‘tool-kit’ of structurally-related solubilising polymers, with each providing different advantages for studying membrane proteins encapsulated in lipid particles.

One of the central themes of biomolecular engineering is the challenge of exploiting the properties of biological materials. Part of this challenge has been uncovering and harnessing properties of biological components that only emerge following their ordered self-assembly. One biomolecular building block that has received significant interest in the past decade is the M13 bacteriophage. There have been a number of recent attempts to trigger the ordered assembly of M13 bacteriophage into multivirion structures, relying on the innate tendency of M13 to form liquid crystals at high concentrations. These, in general, yield planar two-dimensional materials. Presented here is the production of multivirion assemblies of M13 bacteriophage via the chemical modification of its surface by the covalent attachment of the xanthene-based dye tetramethylrhodamine (TMR) isothiocyanate (TRITC). We show that TMR induces the formation of three-dimensional aster-like assemblies of M13 by providing “adhesive” action between bacteriophage particles through the formation of H-aggregates (face-to-face stacking of dye molecules). We also show that the H-aggregation of TMR is greatly enhanced by covalent attachment to M13 and is enhanced further still upon the ordered self-assembly of M13, leading to the suggestion that M13 could be used to promote the self-assembly of dyes that form J-aggregates, a desirable arrangement of fluorescent dye, which has interesting optical properties and potential applications in the fields of medicine and light harvesting technology.