Characterization of D1 Dopamine Receptor Posttranscriptional Regulation
Posttranscriptional regulation (PTR) of gene expression describes regulatory mechanisms that control the expression of protein from its cognate mRNA. Studies that investigate changes in gene expression, for a variety of reasons, typically focus on measuring levels of mRNA or protein but not both. Even studies that measure both mRNA and protein levels of the gene of interest rarely assess the temporal discordance between the two. Given that PTR provides a mechanism for spatial and temporal regulation of gene expression, it likely plays a major role in physiological and pathophysiological conditions. In this chapter, we describe methods to assess PTR using the D1 dopamine receptor gene as an example. PTR mechanisms can be broadly classified into mechanisms that regulate mRNA turnover and those that control mRNA translation. The mouse catecholaminergic CAD cell line which expresses endogenous D1 dopamine receptor is a tractable model system for deciphering the molecular mechanism of D1 receptor PTR. We describe methods to measure D1 dopamine receptor mRNA stability using actinomycin D and methods using reporter constructs to assess microRNA (miRNA)-mediated regulation of D1 receptor protein translation. Using these methods we demonstrate that the D1 dopamine receptor exhibits PTR in which the expression of D1 receptor protein is regulated by miRNAs. The chapter provides detailed methods for studying potential D1 dopamine receptor PTR during development and in disease states.
Key wordsDopamine receptor Gene expression Posttranscriptional regulation mRNA stability mRNA translation MicroRNA 3′ untranslated region RT-PCR Western blotting
The protocols described here were developed with the help of Jennifer Pasuit, Dr. Denis Chang, and Dr. Thuy Do. Funding was provided by the F.M. Kirby Foundation, the UMDNJ Foundation, and NIH grant (DA0260300)
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