Characterization of D1 Dopamine Receptor Posttranscriptional Regulation

  • Eldo V. KuzhikandathilEmail author
Part of the Neuromethods book series (NM, volume 96)


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 words

Dopamine 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)


  1. 1.
    Yoo S, van Niekerk EA, Merianda TT, Twiss JL (2010) Dynamics of axonal mRNA transport and implications for peripheral nerve regeneration. Exp Neurol 223(1):19–27PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Willis DE, van Niekerk EA, Sasaki Y, Mesngon M, Merianda TT, Williams GG, Kendall M, Smith DS, Bassell GJ, Twiss JL (2007) Extracellular stimuli specifically regulate localized levels of individual neuronal mRNAs. J Cell Biol 178(6):965–980PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Schambra UB, Duncan GE, Breese GR, Fornaretto MG, Caron MG et al (1994) Ontogeny of D1A and D2 dopamine receptor subtypes in rat brain using in situ hybridization and receptor binding. Neuroscience 62(1):65–85PubMedCrossRefGoogle Scholar
  4. 4.
    Jung AB, Bennett JP (1996) Development of striatal dopaminergic function. I. Pre- and postnatal development of mRNAs and binding sites for striatal D1 (D1a) and D2 (D2a) receptors. Brain Res Dev Brain Res 94(2):109–120PubMedCrossRefGoogle Scholar
  5. 5.
    Tobón KE, Chang D, Kuzhikandathil EV (2012) MicroRNA 142-3p mediates post-transcriptional regulation of D1 dopamine receptor expression. PLoS One 7(11):e49288Google Scholar
  6. 6.
    Brana C, Caille I, Pellevoisin C, Charron G, Aubert I et al (1996) Ontogeny of the striatal neurons expressing the D1 dopamine receptor in humans. J Comp Neurol 370(1):23–34PubMedCrossRefGoogle Scholar
  7. 7.
    Pasuit JB, Li Z, Kuzhikandathil EV (2004) Multi-modal regulation of endogenous D1 dopamine receptor expression and function in the CAD catecholaminergic cell line. J Neurochem 89(6):1508–1519PubMedCrossRefGoogle Scholar
  8. 8.
    Do T, Kerr B, Kuzhikandathil EV (2007) Brain-derived neurotrophic factor regulates the expression of D1 dopamine receptors. J Neurochem 100(2):416–428PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Pharmacology and PhysiologyRutgers-New Jersey Medical SchoolNewarkUSA

Personalised recommendations