The structure and function of the beta chemokine receptor D6 - a member of the family of G protein coupled receptors

Receptors are proteins embedded in cell membranes with different parts of the molecule extending above and below the membrane surface. When a receptor is bound by its specific ligand on the exterior of the cell, a signal is transmitted to the interior, setting up a chain of events that determine how the cell responds to the signal. Examples are the response of cells when hormones bind to their receptors, or the olefactory response triggered when odour molecules bind receptors in the nose. Since they control the way in which cells respond to external signals, receptors are vitally important in maintaining the well being of the cell. Drugs can bind to receptors and, by their response, influence the biology of the cell. More than half of the drugs in present use interact with receptors. There is a large number of receptors that all have a common structure and similar initial chemical responses to external stimuli. Members of this so-called G-Protein Coupled Receptor (GPCR) family are found acting in situations as diverse as the recognition of taste and odour, responses to tissue damage, mood and behaviour and some cancers. Despite their importance, there is very little knowledge of the three-dimensional structures of GPCRs, owing to the extreme difficulty in extracting the receptors from cells in large enough quantities to purify and crystallise for structural studies. Some years ago we discovered a GPCR, called D6, that binds chemokines (small proteins that are produced in response to tissue damage and stimulate inflammatory responses) and can be produced in relatively large amounts. We can extract and purify D6 and have found a way to grow crystals of D6. We can now determine its 3-dimensional structure using a technique called x-ray crystallography. Once we know the structure of D6 we will be better able to understand how it functions and to design drugs that adapt its biological behaviour. Furthermore, because of the similarity of D6 with other GPCRs, the structure will be very useful as a model for other receptors and provide new insights for further research.

Because of their wide range of biological functions, member of the large GPCR family of integral membrane protein receptors are of special interest in biological, biochemical, medical and pharmaceutical research. These receptors share a common structural fold of seven transmembrane helices. To date the only such receptor to have its crystal structure elucidated is rhodopsin and structures of both bacterial and mammalian forms have been determined. Valuable though these structures are, they are of limited use in helping develop methods for structure-based rational anti-GPCR drug design. Recently, with BBSRC support, we have been studying D6, a 7-TM GPCR. D6 is a member of small population of atypical chemokine receptors that are apparently signalling-incompetent and are characterised by a promiscuous interaction with diverse chemokine ligands. We have an overexpression system in a mammalian cell line that provides mg quantities of purified D6 and have shown that the receptor is glycosylated, sulphated and constitutively phosphorylated. We have prepared a non-glycosylated mutant with ligand binding characteristics and expression levels identical to the wild type receptor. We have demonstrated that the relatively large number of D6 receptors is due to the receptor being internalised and recycled. We have isolated and purified D6 and have obtained crystals that diffract X-rays to a resolution of 8 Angstroms. We intend to continue this work, which is ultimately aimed at obtaining a structure for the receptor and will (i) characterise the crystallised protein and refine the crystallisation conditions to obtain crystals diffracting to sufficient resolution to provide a structure; (ii) determine the structure using protein crystallography techniques; (iii) prepare, crystallise and obtain the structure of a receptor/ligand complex; (iv) investigate the details of ligand binding; and (v) investigate the significance of the structure as a model for other GPCRs.