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A negative regulatory element in the promoter region of the rat alpha 2A-adrenergic receptor gene overlaps an SP1 consensus binding site.

ABSTRACT: Three subtypes of alpha 2-adrenergic receptors (alpha 2A, alpha 2B and alpha 2C) have been described that differ in their primary sequence and tissue-specific expression and are encoded by three distinct genes. Previous work has shown that the human alpha 2A-adrenergic receptor gene promoter consists of a TATA-box (TATAAA), palindromic sequence (CCCACGTGGG) and GC-box (GGGGCGG) motif. Sequence analysis of the putative promoter region of the rat alpha 2A-adrenergic receptor gene showed that these promoter regions are conserved in their sequence and relative location. We analysed the transcriptional activity of these regions using RINm5F, a rat insulinoma cell line that expresses the endogenous alpha 2A-adrenergic receptor gene. These results showed that the region from -484 to -92 has a negative effect on transcription, as deletion of this region in alpha 2A-adrenergic receptor gene-chloramphenicol acetyltransferase reporter constructs increased reporter gene activity. This region included the GC-box sequence which is a consensus binding site for the nuclear factor SP1, which is a positive activator of transcription. Gel-mobility-shift assays and supershift assays with an antibody that recognizes SP1 showed binding of the SP1 nuclear factor as well as other nuclear factors to this GC-box region. Additional nuclear factors bind to the downstream palindromic region. We suggest that positive- and negative-acting nuclear factors contribute to the activity of the alpha 2-adrenergic receptor promoter.

SUBMITTER: Handy DE

PROVIDER: S-EPMC1136033 | biostudies-other | 1995 Oct

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Type 2A protein phosphatase (PP2A) enzymes are serine/threonine phosphatases which comprise a scaffold A subunit, a regulatory B subunit and a catalytic C subunit, and have been implicated in the dephosphorylation of multiple cardiac phosphoproteins. B subunits determine subcellular targeting, substrate specificity and catalytic activity, and can themselves be regulated by post-translational modifications. We explored potential ?-adrenergic regulation of PP2A in cardiomyocytes through phosphorylation of the regulatory B subunit isoform B56?. METHODS AND RESULTS:Phosphate affinity SDS-PAGE and immunoblot analysis revealed increased phosphorylation of B56? in adult rat ventricular myocytes (ARVM) exposed to the ?-adrenergic receptor (?AR) agonist isoprenaline (ISO). Phosphorylation of B56? occurred at S573, primarily through stimulation of the ?1AR subtype, and was dependent on PKA activity. The functional role of the phosphorylation was explored in ARVM transduced with adenoviruses expressing wild type (WT) or non-phosphorylatable (S573A) B56?, fused to GFP at the N-terminus. C subunit expression was increased in ARVM expressing GFP-B56?-WT or GFP-B56?-S573A, both of which co-immunoprecipitated with endogenous C and A subunits. PP2A activity in cell lysates was increased in response to ISO in ARVM expressing GFP-B56?-WT but not GFP-B56?-S573A. Immunoblot analysis of the phosphoproteome in ARVM expressing GFP-B56?-WT or GFP-B56?-S573A with antibodies detecting (i) phospho-serine/threonine residues in distinct kinase substrate motifs or (ii) specific phosphorylated residues of functional importance in selected proteins revealed a comparable phosphorylation profile in the absence or presence of ISO stimulation. CONCLUSIONS:In cardiomyocytes, ?AR stimulation induces PKA-mediated phosphorylation of the PP2A regulatory subunit isoform B56? at S573, which increases associated PP2A catalytic activity. This is likely to regulate the phosphorylation status of specific B56?-PP2A substrates, which remain to be identified.

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A negative regulatory element in the promoter region of the rat alpha 2A-adrenergic receptor gene overlaps an SP1 consensus binding site.

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