Related Experiment Video
Updated: Jun 25, 2026

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators
Published on: February 20, 2018
Regulation of cocaine- and amphetamine-regulated transcript mRNA expression by calcium-mediated signaling in GH3
D C Jones1, A Lakatos, G A Rogge
1Division of Neuroscience, Yerkes National Primate Research Center of Emory University, 945 Gatewood Road, Atlanta, GA 30329, USA. douglas.jones@emory.edu
Abstract:
Cocaine- and amphetamine-regulated-transcript (CART) peptides are associated with multiple physiological processes, including, feeding, body weight, and the response to drugs of abuse. CART mRNA and peptide levels and the expression of the CART gene appears to be under the control of a number of extra- and intra-cellular factors including the transcription factor, cAMP response element binding protein (CREB). Similar to the effects of CART, Ca(2+) signaling leads to the phosphorylation of CREB and has been associated with both feeding and the actions of psychostimulants; therefore, we hypothesized that Ca(2+) may play a role in CART gene regulation. We used real-time PCR (rtPCR) and GH3 cells to examine the effect of ionomycin, which increases intracellular Ca(2+), on CART mRNA levels. Ionomycin increased CART mRNA in a dose- and time-dependent manner. The effect of ionomycin appeared transient as CART mRNA had returned to control levels 3 h following treatment. Calmidazolium and KN93, inhibitors of calmodulin and Ca(2+)-modulated protein (CaM) kinases respectively, attenuated the effect of ionomycin (10 microM) on CART mRNA levels suggesting a calmodulin-dependent mechanism. Western immunoblotting indicated that ionomycin increased phosphorylated cAMP response element binding protein (pCREB) levels and electrophoretic mobility shift assay/supershift assay using antibodies against pCREB demonstrated increased levels of a CART oligo/pCREB protein complex. Finally, we showed that injection of ionomycin into the rat nucleus accumbens increases CART mRNA levels. To our knowledge, this is the first study providing evidence that the CART gene is, in part, regulated by Ca(2+)/CaM/CREB-dependent cell signaling.
Related Concept Videos
GPCRs Regulate Adenylyl Cylase Activity
Two...
Feedback Regulation of Calcium Concentration
Various transmembrane receptors, such as G protein-coupled receptors (GPCRs), elicit a response to extracellular signals by increasing cytosolic calcium. Activated GPCRs...
Calmodulin-dependent Signaling
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...
Cell Specific Gene Expression
cAMP-dependent Protein Kinase Pathways
G-Protein Gated Ion Channels
Sensory organs,...

