The significance of Ser1029 of the heat-stable enterotoxin receptor (STaR): relation of STa-mediated guanylyl cyclase activation and signaling by phorbol myristate acetate.

To characterize Ser1029 in STaR at a consensus sequence of phosphorylation site by PKC, two mutants of mS1029A with replacement of Ser1029 to Ala1029 and C delta 1029 lacking 22 amino acids including Ser1029 were prepared. Preincubation of the wild type-STaR (wt-STaR) transfectant with 1 microM PMA caused additional STa-mediated guanylyl cyclase (GC) activation compared to control, whereas the mS1029A- and C delta 1029-transfected cells did not show a similar enhanced GC activation by PMA. After metabolic labeling with [32P]phosphate, transfected cells with wt-STaR and mutants were incubated with 1 microM PMA. Subsequent 32P-radiolabeled proteins were immunoprecipitated using anti-STaR antibody, and analyzed by autoradiography after separation on SDS-PAGE. The immunoprecipitated wt-STaR but not mS1029A and C delta 1029 had a significant radioactivity. These results suggest that the effect of PMA on wt-STaR transfectants may be caused by phosphorylation of Ser1029. The C delta 1012 mutant, with further truncation (Gln1012-Phe1050) of the carboxy terminus, did not show STa-mediated GC activation. Based on these data, these 17 amino acids (Gln1012-Ala1028), essential for signaling of GC activation by STa, have an abundance of basic amino acids which might be functionally influenced by phosphorylation of Ser1029.