G-protein-coupled receptors (GPCRs) are of prime importance for cell signal transduction mechanisms and are the target of many current and potential drugs. However, structural data on these membrane proteins is still scarce because of their low natural abundance and the low efficiency of most of the expression systems currently available. This review presents the most important expression systems currently employed for heterologous expression of GPCRs; Escherichia coli, yeast, insect cells and mammalian cells. After briefly recalling the specificity, advantages and limitations of each system, particular emphasis is put on the quantitative comparison of these expression systems in terms of overall expression yield, and on the influence of various factors (primary sequence, origin, cell type, N- and C-terminal tags) on the results.

Rhizobium species strain NGR234 is the most promiscuous known rhizobium. In addition to the non-legume Parasponia andersonii, it nodulates at least 70 genera of legumes. Here we show that the nodulation genes of this bacterium determine the production of a large family of Nod-factors which are N-acylated chitin pentamers carrying a variety of substituents. The terminal non-reducing glucosamine is N-acylated with vaccenic or palmitic acids, is N-methylated, and carries varying numbers of carbamoyl groups. The reducing N-acetyl-glucosamine residue is substituted on position 6 with 2-O-methyl-L-fucose which may be acetylated or sulphated or non-substituted. All three internal residues are N-acetylated. At pico- to nanomolar concentrations, these signal molecules exhibit biological activities on the tropical legumes Macroptilium and Vigna (Phaseoleae), as well as on both the temperate genera Medicago (Trifoliae) and Vicia (Viciae). These data strongly suggest that the uniquely broad host range of NGR234 is mediated by the synthesis of a family of varied sulphated and non-sulphated lipo-oligosaccharide signals.

Although Rhizobium sp. NGR234 and Rhizobium fredii USDA257 share many traits, dysfunctional nodSU genes in the latter prohibit nodulation of Leucaena species. Accordingly, we used R. fredii transconjugants harboring the nodS and nodU genes of NGR234 to study their role in the structural modification of the lipo-oligosaccharide Nod factors. Differences between the Nod factors mainly concern the length of the oligomer (three to five glucosamine residues in USDA257 and five residues only in NGR234) and the presence of additional substituents in NGR234 (N-linked methyl, one or two carbamoyl groups on the non-reducing moiety, acetyl or sulfate groups on the fucose). R. fredii(nodS) transconjugants produce chitopentamer Nod factors with a Nlinked methyl group on the glucosaminyl terminus. Introduction of nodU into USDA257 results in the formation of 6-O-carbamoylated factors. Co-transfer of nodSU directs N-methylation, mono-6-O-carbamoylation, and production of pentameric Nod factors. Mutation of nodU in NGR234 suppresses the formation of bis-carbamoylated species. Insertional mutagenesis of nodSU drastically decreases Nod factor production, but with the exception of sulfated factors (which are partially N-methylated and mono-carbamoylated), they are identical to those of the wild-type strain. Thus, Nod factor levels, their degree of oligomerization, and N-methylation are linked to the activity encoded by nodS.

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The heterotrophic and mesophilic marine bacterium HYD-1545 was isolated on a metal-amended medium from the dorsal integument of the hydrothermal vent polychaete Alvinella pompejana. This strain, which can be assigned to the genus Alteromonas on the basis of its G+C content and phenotypical features, produced large amounts of an acidic polysaccharide in batch cultures. The polysaccharide was excreted during the stationary phase of growth and contained glucose, galactose, glucuronic acid, galacturonic acid, and 4,6-O-(1-carboxyethilidene)-galactose as major components. This polysaccharide was a polyelectrolyte, and the viscosity of its solutions depended on the ionic strength. The decrease in viscosity with increasing NaCl concentrations and the effect of Ca21 in decreasing the viscosity at low Ca2+ concentrations support a model in which the polysaccharide carries anionic groups. However, an unusual behavior was observed at higher concentrations and could be related to intermolecular interactions involving Ca2+ ions.

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G protein-coupled receptors (GPCRS) represent a class of integral membrane proteins involved in many biological processes and pathologies. Fifty percent of all modern drugs and almost 25% of the top 200 bestselling drugs are estimated to target GPCRs. Despite these crucial biological implications, very little is known, at atomic resolution, about the detailed molecular mechanisms by which these membrane proteins are able to recognize their extra-cellular stimuli and transmit the associated messages. Obviously, our understanding of GPCR functioning would be greatly facilitated by the availability of high-resolution three-dimensional (3D) structural data. However, expression, solubilization and purification of these membrane proteins are not easy to achieve, and at present, only one 3D structure has been determined, that of bovine rhodopsin. This review presents and compares the different successful strategies which have been applied to solubilize and purify recombinant GPCRs in the perspective of structural biology experiments.