| Abstrakt: |
RGS3 is the largest member of a recently discovered family of proteins (RGS proteins) that appear to function as negative regulators of heterotrimeric G-protein signaling. Seventeen mammalian RGS proteins have been identified by cloning or by comparison to expressed sequence tags, and several of these proteins have been shown recently to function as GTPase-activating proteins for G-protein α subunits. Despite the intense interest in RGS proteins as physiological regulators of G-protein signaling, there is little understanding of the structure and regulation of mammalianRGSgenes. Using long-distance PCR, we amplified and characterized the entire coding and 5′-untranslated region of the humanRGS3gene. The coding region of the humanRGS3gene spans 14.7 kb and contains six exons, and the 5′-untranslated region spans 3.2 kb and contains two exons. Mapping of the exons revealed that the RGS domain, conserved among all RGS proteins, was encoded by three exons, while the unique amino-terminal domain of RGS3 was encoded by a single exon. Comparison of the location of the intron–exon boundaries of the humanRGS3gene to that of the humanRGS2gene, the only mammalian RGS gene described previously, revealed a remarkable similarity, providing the first conceptual support for a common ancestral mammalianRGSgene. 5′-RACE analysis was used to map the transcription start site 517 bp upstream of the translation start site, and anchored PCR was performed to amplify 1.0 kb of genomic DNA upstream of the transcription start site. Analysis of the 5′-flanking region revealed the presence of many potential regulatory elements, the presence of an initiator (Inr) element overlapping the transcription start site, and the absence of a TATA or a CCAAT box at the usual positions. By radiation hybrid mapping, theRGS3gene was assigned to human chromosome 9q31–q33. This study is the first to elucidate the structure, chromosomal location, and regulatory sequences of theRGS3gene, and it establishes the genetic basis forRGS3gene research in humans. |
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