Characterization of an epitope-tagged guanylyl cyclase-A receptor

Abstract

Background In some forms of arterial hypertension and as one of the earliest and pathognomonic events in cardiac hypertrophy and insufficiency, the cardiac synthesis and release of ANP and BNP is markedly enhanced, but the GC-A-mediated effects of the NPs are clearly diminished, indicating a receptor or postreceptor defect of GC-A. Thus, from a pathophysiological perspective, identifying the specific mechanisms involved in the downregulation of GC-A activity has important implications. Biochemical studies in transfected GC-A-overexpressing cells showed that phosphorylation of GC-A is essential for its activation process. In turn, inactivation of GC-A probably involves dephosphorylation [1]. NP-dependent activation of GC-A can be reduced not only by chronic exposure to NPs (homologous desensitization) but also by exposure to agents other than NPs (heterologous desensitization). It has been shown in cell culture systems that Angiotensin II and endothelin decrease the responsiveness of GC-A. This is probably mediated by a protein kinase C-induced dephosphorylation of GC-A. However, ultimately the precise posttranslational modifications leading to homologous vs. heterologous GC-A desensitization have not been fully characterized. The present study describes the functional characterization of an epitope-tagged GC-A receptor which might help to enrich the receptor for the characterization of putative posttranslational modifications. Methods, results and conclusion The FLAG (DYKDDDDK) epitope was positioned immediately after the cleavage site of the signal peptide by PCRmediated mutagenesis (pCMV5-FLAG-GC-A) [2]. HEK cells were transfected with cDNAs for GC-A or GC-A tagged with the FLAG epitope.