title: Co-expression of the neurokinin NK2 receptor and G-protein components in the fission yeast Schizosaccharomyces pombe.
abstract: The fission yeast Schizosaccharomyces pombe has proven useful for studying molecular interactions between a range of signal transduction components. We now report the first co-expression of a mammalian seven-transmembrane receptor and G-protein components in S. pombe. We selected the human neurokinin NK2 receptor together with its G-protein-signalling partner Gq for this study. Yeast membrane fractions showed high levels of NK2 receptor-binding activity (1159 +/- 534 (n = 3) fmol/mg protein) although initial experiments with intact cells revealed an absence of receptors at the cell surface. Using a construct comprising the NK2 coding sequence fused with the signal sequence from an endogenous phosphatase (phoI), we detected approximately 400 NK2 receptors/cell in unbroken yeast. Successful co-expression of the NK2 receptor with the G-protein subunits G alpha q, beta 1 or beta 2 and gamma 3 failed to modulate agonist binding, suggesting the absence of functional interaction between these components. As an alternative test of G alpha q function, we next expressed its downstream effector target phospholipase C-beta 1 (PLC beta 1) in S. pombe. Although PLC beta 1 undergoes powerful in vitro activation by G alpha q derived from baculovirus-infected Sf9 cells and mammalian cells, G alpha q expressed in S. pombe is totally ineffective. Similar results were also achieved with the G-protein subunit G alpha 16. Together, these data suggest that seven-transmembrane receptors can be expressed in S. pombe at high levels and directed to the cell surface although their interaction with co-expressed G-proteins in undetectable. Production of inactive G alpha-chains in S. pombe may account for these observations.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001549:Benzamides / Q000378:metabolism; D001667:Binding, Competitive; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000096:biosynthesis* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008322:Mammals; D051379:Mice; D008969:Molecular Sequence Data; D015288:Neurokinin A / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D010446:Peptide Fragments / Q000276:immunology; D010880:Piperidines / Q000378:metabolism; D018041:Receptors, Neurokinin-2 / Q000096:biosynthesis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012568:Schizosaccharomyces / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D000906:Antibodies; D001549:Benzamides; D009842:Oligopeptides; D010446:Peptide Fragments; D010880:Piperidines; D018041:Receptors, Neurokinin-2; D011994:Recombinant Proteins; C088270:GR 94800; C073839:SR 48968; D015288:Neurokinin A; D019204:GTP-Binding Proteins