Invited Symposium: Angiotensin Receptors
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Krishnamurthi, K (Department of Physiology, Georgetown University Medical Center, USA)
Verbalis, AD (Department of Medicine, Georgetown University Medical Center, USA)
Mok, K-Y (Department of Physiology, Georgetown University Medical Center, USA)
Mulroney, S (Department of Physiology, Georgetown University Medical Center, USA)
Abstract
RNA binding proteins (BPs) were identified in the kidney cortex which bind to cis elements within the 5' leader sequence (5'LS) of the rat type 1a angiotensin (AT1a) receptor mRNA. 5'LS BP activities were previously shown to decrease by 2-fold when AT1 receptor number was doubled in remnant kidneys after compensatory renal growth (CRG). This reciprocal regulation of 5'LS BP activities and AT1 receptor expression led us to investigate the hypothesis that 5'LS BPs inhibit translation of the AT1 receptor. The 5'LS of the rat AT1a receptor was cloned upstream from luciferase in the pSP-luc vector (Promega) in both the sense (S-5'LS-luc) and antisense (AS-5'LS-luc) directions. The AS-5'LS-luc serves as a control construct since we found in gel retention assays that antisense 5'LS RNA (IC50 > 50 nM) was a poor competitor of 5'LS-protein complex formation in marked contrast to sense 5'LS RNA (IC50 ª 2 nM). In vitro translation assays of S-5'LS-luc and AS-5'LS-luc demonstrated that BPs (3 µg) selectively inhibited translation of luciferase in the S-5'LS-luc construct (35 ± 7%) compared with AS-5'LS-luc (92 ± 12%) with respect to controls (defined as the incorporation of 35S-methionine into luciferase in the absence of BPs). Under these conditions, translation of luciferase in the S-5'LS-luc construct in the presence of sense 5'LS RNA (20nM) resulted in abrogation of the BP-induced inhibition [% Control: 5'LS without BP, 95±11%; BP alone, 35 ± 7%; 5'LS + BP, 105±12%], indicating that 5'LS RNA can compete with S-5'LS-luc for BP. In conclusion, these results suggest that 1) AT1 receptor translation is inhibited by renal cytosolic proteins which bind to the 5'LS of the receptor RNA and 2) physiological regulation of 5'LS BP activities play a significant role in mediating changes in AT1 receptor gene expression during CRG. Supported by NKF-DC Affiliate.
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Presentation Number SAsandberg0492
Keywords: angiotensin, AT1 receptor, kidney, translation, binding protein
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