COEXPRESSION OF THE NEUROKININ NK2 RECEPTOR AND G-PROTEIN COMPONENTS IN THE FISSION YEAST SCHIZOSACCHAROMYCES-POMBE

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 G(q) 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 (pho1), we detected similar to 400 NK2 receptors/cell in unbroken yeast. Successful coexpression 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,vith 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.