Characterization of RNA-binding proteins possibly involved in modulating human AT 1 receptor mRNA stability

Abstract

Angiotensin II exerts its cardiovascular effects mainly through the activation of AT(1) receptors. These receptors can be regulated at a post-transcriptional level, that is through the modulation of the mRNA stability. This regulation usually involves proteins which are able to bind the 3'UTR of the mRNA molecule. The experiments of the present paper were performed in order to characterize the RNA-binding proteins interacting with the hAT(1) receptor mRNA in human vascular smooth muscle cells. Immunoblot analysis allowed us to identify three different RNA-binding proteins, AUF1, HuR, and hnRNP A1. UV cross-linking and immunoprecipitation experiments demonstrated that AUF1 binds to the hAT(1)-receptor mRNA radiolabeled probes specifically, but in different ways in relation to the clinically important A/C gene polymorphism. Gel shift experiments using purified recombinant proteins confirmed the specificity of interaction of these proteins with the hAT(1)-receptor mRNA. In basal conditions the proteins were mainly located in the nuclei, but angiotensin II administration clearly induced their translocation to the cytosol. This observation was confirmed by transfection experiments using both GFP/AUF1 and GFP/HuR fusion proteins. Our findings allow identification of specific RNA-binding proteins possibly involved in the control of the hAT1-receptor mRNA stability and in the regulation of their expressions.