Poster Presentation Melbourne Protein Group Student Symposium 2013

The Small Interacting Protein, CRIP1a, is a Novel Modulator of Cannabinoid CB1 Receptor Mediated Signalling (#40)

Nilushi S Karunaratne 1 , Paul J White 1 , Mark Connor 2 , Daniel T Malone 1
  1. Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia
  2. The Australian School of Advanced Medicine, Macquarie University, North Ryde, NSW, Australia

Introduction: The CB1 receptor, a highly abundant GPCR in the CNS, has been targeted therapeutically for seizures, nausea, depression and addiction. However, CB1 compounds have had limited clinical use due to their side effect profiles. Cannabinoid receptor interacting protein 1a (CRIP1a) is a recently discovered intracellular protein that interacts with the C-terminal tail of the CB1 receptor. Preliminary studies suggest that CRIP1a has inhibitory effects on CB1 receptor function. This study aimed to determine whether measurement of membrane potential in intact cells is a suitable assay for examining changes in CB1 signalling. Secondly, to investigate the effect of decreased expression of CRIP1a on a specific downstream CB1 receptor mediated effect; activation of G-protein coupled inwardly rectifying potassium (GIRK) channels. Method: Measurement of membrane potential was used to examine changes in CB1 signalling. Results: The CB1 receptor agonist WIN55212-2 produced a maximum change in fluorescence of 30.54±2.31% (-log EC50, 6.80) whilst CP55940 produced a maximum change in fluorescence of 27.09±1.34% (-log EC50, 7.12). Anandamide as expected showed both a decrease in efficacy (22.52±1.00) and potency (-log EC50, 5.68) at the CB1 receptor. To achieve decreased CRIP1a expression, initial studies screened three individual CRIP1a-siRNA (n=3) in AtT20 cells inherently expressing CRIP1a and stably expressing CB1 receptors. Western blot analysis of cells transfected with CRIP1a-siRNA (5nM, 20nM and 40nM) showed all CRIP1a-siRNAs to induce significant reduction in CRIP1a protein levels (p < 0.001). This siRNA-induced CRIP1a knockdown significantly increased anandamide-induced K+ channel activation (p < 0.05) in AtT20 cells whilst no change was observed with WIN55212-2 and CP55940. Pre-treating cells with CB1 antagonist AM251 prior to CB1 receptor agonist administration confirmed these effects to be CB1 mediated. Proteins that interact with GPCRs provide a new avenue for modulation and fine-tuning receptor activity. This research could lead to the development of new drugs that have therapeutic benefits in conditions where CB1 receptor signalling is altered or off-balance such as schizophrenia, depression and anxiety-like disorders.

  1. Niehaus J et al. (2007), Molecular Pharmacology vol. 72, (6) 1557-1566
  2. Stauffer et al. (2011), Neuroscience Letters 2011 vol. 503, (3) 224-228