The effect of H pylori eradication on symptom relief in patients with functional dyspepsia

Abstract

• AGONIST-STIMULATED PHOSPHORYLATION OF THE CARBOXYLTERMINAL TAIL OF THE SECRETIN RECEPTOR. F. 0zcelebi, M. Holtmann, R. Rentsch, L.J. Miller. GI Basic Research Unit, Mayo Clinic, Rochester, MN 55905 The secretin receptor belongs to a distinct family of receptors in the G protein-coupled receptor superfamily. The absence of sequence motifs typical of the B-adrenergic receptor family in which regulatory mechanisms have been extensively studied, raises questions of the comparability of regulation of the secretin receptor family. In this work, we have focused on a key mechanism of receptor desensitization, receptor phosphorylation, in cell fines expressing the rat secretin receptor. We have studied receptor phosphorylation in intact transiently transfected COS cells and a stable receptor-bearing CHO cell line, utilizing a strategy we described for the acinar cell CCK receptor (Klueppelberg et al, J. Biol. Chem. 1991). Indeed, this approach resulted in the demonstration of agonist-stimulated receptor phosphorylation. Secretin phosphoreceptor migrated on a SDS-polyacrylamide gel at M,=57,000-62,000 in its native state, and at M,=42,000 after deglycosylation. This is identical to electrophoretic behavior of the affinity-labeled receptor. Phosphorylation occurred rapidly in a secretagogue concentration-dependent manner, with I#M secretin eliciting a 7.2fold increase in phosphorylation after 2 min. One-dimensional phosphopeptide mapping after cyanogen bromide cleavage revealed a single phosphopeptide band of approximate M,= 9,400, corresponding to the expected size of the carboxylterminal tail of the receptor. This identification was then confirmed with a truncation mutant in which all potential sites of phosphorylation in that domain were eliminated, and no agonist-stimulated phosphorylation was observed. Phosphoamino acid analysis of the secretin phosphoreceptor demonstrated predominance of phosphothreonine over phosphoserine (3.2:1), with no phosphotyrosine observed. Three distinct carboxyl-terminal truncation mutants were constructed to each eliminate a subset of potential phosphorylation sites. Appropriate biosynthetic processing, expression on the cell surface, ligand binding, and signalling were assured by demonstration of affinity labeling and cAMP responsiveness. Differential levels of phosphorylation provide additional insights into the distinct residues phosphorylated on this receptor molecule. In conclusion, this work demonstrates for the first time that receptors in the newlydescribed secretin receptor family are phosphorylated in response to agonist stimulation. The molecular details of secretin receptor phosphorylation are highly analogous to those of the /3-adrenergic receptor family. This suggests that phosphorylation is also likely to represent an important molecular mechanism for receptor desensitization for this family of receptors. THE EFFECT OF H PYLOR[ ERADICATION ON SYMPTOM RELIEF IN PATIENTS WITH FUNCTIONAL DYSPEPSIA. MM Ozmen, RS Patankar, CD Johnson