Dopamine-1A (D1A) receptor gene transcription in renal cells: Evidence for novel CRE-like and 5-prime-UTR intronic elements.
Item
-
Title
-
Dopamine-1A (D1A) receptor gene transcription in renal cells: Evidence for novel CRE-like and 5-prime-UTR intronic elements.
-
Identifier
-
AAI9720125
-
identifier
-
9720125
-
Creator
-
O'Rourke, Dawn A.
-
Contributor
-
Adviser: Dennis P. Helay
-
Date
-
1997
-
Language
-
English
-
Publisher
-
City University of New York.
-
Subject
-
Health Sciences, Pharmacology | Biology, Molecular
-
Abstract
-
The purpose of this study was to characterize the dopamine-1A (D{dollar}\sb{lcub}\rm 1A{rcub}{dollar}) receptor gene promoter in renal epithelial cells. D{dollar}\sb{lcub}\rm 1A{rcub}{dollar} receptor gene transcription was elevated in LLC-PK{dollar}\sb1{dollar} cells following short term exposure to dopamine, as D{dollar}\sb{lcub}\rm 1A{rcub}{dollar} receptor mRNA levels were increased D{dollar}\sb{lcub}\rm 1A{rcub}{dollar} receptor mRNA stability was decreased. Transcriptional regulation of the D{dollar}\sb{lcub}\rm 1A{rcub}{dollar} receptor gene was further examined using constructs containing progressive deletions in the D{dollar}\sb{lcub}\rm 1A{rcub}{dollar} 5{dollar}\sp\prime{dollar}-flanking region ({dollar}-{dollar}3kb to +53) placed upstream of a promoter-less chloramphenicol acetyltransferase reporter gene (pCAT). The highest activity ({dollar}>{dollar}50 fold) in LLC-PK{dollar}\sb1{dollar} cells was isolated to a construct which extended to {dollar}-{dollar}303. DNase I analysis and gel mobility shift assays indicated that several consensus sites (AP2, CRE-like, E-box, GC-box, Sp1/Egr-1) within {dollar}-{dollar}240 to {dollar}-{dollar}40 of the D{dollar}\sb{lcub}\rm 1A{rcub}{dollar} receptor gene bound LLC-PK{dollar}\sb1{dollar} nuclear extracts.;The CAT activity of pCAT-303 increased 2-fold in response to increased intracellular cAMP. The {dollar}-{dollar}303 to +962 sequence contains two cAMP response-like elements (CRE) separated by 3 bases: 5{dollar}\sp\prime{dollar}-CRE, AGACGTCA, and 3{dollar}\sp\prime{dollar}-CRE; GGACGTCC. The 3{dollar}\sp\prime{dollar}-CRE is better conserved across species and a single base substitution within the 3{dollar}\sp\prime{dollar}-CRE reduced the CAT activity of the pCAT-303 construct to twice basal, indicating the 3{dollar}\sp\prime{dollar}-CRE plays a role in basal and inducible expression of the D{dollar}\sb{lcub}\rm 1A{rcub}{dollar} receptor gene. Placement of the 5{dollar}\sp\prime{dollar}- and 3{dollar}\sp\prime{dollar}-CRE sequences in front of a heterologous promoter stimulated CAT activity (nearly 3-fold) independent of orientation.;Attempts to further delineate the porcine D{dollar}\sb{lcub}\rm 1A{rcub}{dollar} receptor activating sequence to regions upstream ({dollar}-{dollar}303/+38) or downstream (+53/+962) of the transcription start site proved neither region was able to independently promote reporter activity. DNase I analysis and gel mobility shift assays demonstrated that LLC-PK{dollar}\sb1{dollar} nuclear proteins could bind to the 3{dollar}\sp\prime{dollar}-intron sequence. Detailed analysis of the 5{dollar}\sp\prime{dollar}-UTR indicated that depending on context the intron can be either a positive or negative element and that an interaction between the 5{dollar}\sp\prime{dollar}-UTR intron and that upstream elements are required for transcriptional activation of the D{dollar}\sb{lcub}\rm 1A{rcub}{dollar} receptor gene promoter in renal cells. This is the first evidence that a G-protein coupled receptor gene is regulated by interactions between elements within a 5{dollar}\sp\prime{dollar}-UTR intron and a promoter.
-
Type
-
dissertation
-
Source
-
PQT Legacy CUNY.xlsx
-
degree
-
Ph.D.
