Cloning and characterization of the GNA11 promoter and its regulation by early growth response 1

Abstract

GNAQ and GNA11, encoding the G-proteins Gα(q) and Gα₁₁, are members of the Gα(q)/Gα₁₁ subfamily, which transmits signals from the cell surface to intracellular signalling cascades. The GNAQ promoter was already characterized, and regulation by the transcription factor early growth response 1 (Egr-1) was demonstrated. Interestingly, in silico analysis revealed putative Egr-1 binding sites in sequences potentially representing the GNA11 promoter. However, the GNA11 promoter has not been characterized so far. Therefore, the purpose of the study was the characterization of the GNA11 promoter and investigation of its potential regulation by Egr-1. The putative GNA11 promoter was cloned, and deletion constructs were generated. Luciferase assays were performed, and essential regulatory regions identified between nt-805/-177. In electrophoretic mobility shift assays (EMSAs), one specific Egr-1 binding site at nt-475/-445 was identified. An Egr-1 expression plasmid was generated, which evoked increased Egr-1 content in nuclear extracts and a > 2-fold increase in GNA11 promoter activity in construct nt-805/+54 (p = 0.035). Finally, real-time PCR analysis was performed, and an increased Gα₁₁ mRNA (p = 0.035) expression induced by Egr-1 was found. Here, we characterize for the first time the GNA11 promoter and its specific interaction with Egr-1. Both the GNAQ and the GNA11 promoter appear to be regulated by the same transcription factor, Egr-1, which may be a molecular mechanism leading to Gα(q)-/Gα₁₁-associated phenotypes.