Submission Number: ANI-4-16-17

Abstract Number: 495

A LUTEINIZING HORMONE RECEPTOR mRNA BINDING PROTEIN FROM RAT OVARY SPECIFICALLY INTERACTS WITH A POLYPYRIMIDINE SEQUENCE IN THE CODING REGION AND CAUSES ACCELERATED DECAY OF THE mRNA.

Anil Nair, Helle Peegel, John Kash and KMJ Menon*

Departments of Obstetrics and Gynecology and Biological Chemistry, University of Michigan, Ann Arbor, MI ¹

Abstract:
Previous studies have shown that in the ovary the loss of steady state levels of LH receptor (LHR) mRNA during hormone-induced down regulation is mediated by accelerated mRNA degradation. By RNA electrophoretic mobility gel shift assay, a 50 kDa ovarian protein, termed LHR mRNA binding protein (LRBP) was shown to be upregulated during ligand - induced down regulation. Site directed mutagenesis and hydroxyl-radical footprinting studies revealed that LRBP binds to a bipartite polypyrimidine sequence5'-U₂₀₃CUCX7UCUCCCU₂₂₀-3' of the LHR mRNA. We report development of an in vitro LHR mRNA decay assay in which the function of the 50 kDa LRBP was tested. Utilizing this in vitro decay system, the effect of purified fraction of LRBP on LHR mRNA decay was examined in reactions containing microsomes isolated from pseudopregnant rat ovaries and exogenously added LHR mRNA. Aliquots of decay reactions were incubated for 0,15,30,60 and 120 min in the absence of added protein, and in the presence of LRBP-1 or BSA. LHR mRNA content was assayed at each interval by northern blot analysis. Half-lives of the exogenous LHR mRNA were calculated from decay reactions utilizing densitometric scans of the 6.7 Kb LHR mRNA transcript. The results show that microsomes incubated in the presence of LRBP degraded LHR mRNA more rapidly than those incubated with BSA or no protein, indicated by 1) the reduction in receptor mRNA half-life (t_1/2=45 min) compared to the controls (t_1/2=70 min) and 2) the complete loss of the 6.7 Kb transcript at 120 minutes. The degradation of LHR mRNA was specific since no degradation of the 18S rRNA was observed. LRBP expression showed a reciprocal relationship with the expression of LHR mRNA during follicle development, ovulation and corpus luteum life span in PMSG-hCG induced rat ovaries. These studies show that LRBP plays an important role in the maintenance of the steady state levels of the LHR mRNA in rat ovaries. .

Keywords: LH receptor, ovary, receptor down-regulation

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This abstract is being presented at: 8:00 AM in session:
Hormone Receptors