Co-expression of the neurokinin NK2 receptor and G-protein components in the fission yeastSchizosaccharomyces 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 coexpression 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 (phol), we detected ∼ 400 NK2 receptors/cell in unbroken yeast. Successful co-expression of the NK2 receptor with the G-protien subunits Gαq, β1 or β2 and γ3 failed to modulate agonist binding, suggesting the absence of functional interaction between these components. As an alternative test of Gαq function, we next expressed its downstream effector target phospholipase C-β1 (PLCβ1) in S. pombe. Al-though PLCβ1 undergoes powerful in vitro activation by Gαq derived from baculovirus-infected Sf9 cells and mammalian cells, Gαq expressed in S. pombe is totally ineffective. Similar results were also achieved with the G-protein subunit Gα16. 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 Gα-chains in S. pombe may account for these observations