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Neuroscience PhD at Wake Forest University at Wake Forest University


Wake Forest University Graduate School » Neuroscience PhD at Wake Forest University

Allyn Howlett, Ph.D.

Allyn Howlett, Ph.D.
Dr. Howlett’s laboratory investigates the mechanisms by which cannabinoid drugs and endocannabinoid compounds stimulate the CB1 cannabinoid receptor to select  intracellular signal transduction pathways.  Cultured neuronal cells are used to define the types of G proteins that interact with the CB1 receptor, and investigate the necessary receptor domains. Phosphorylation of the receptor is an important means to direct signaling through specific G proteins or other signaling proteins such as beta-arrestin, G protein associated sorting protein (GASP) and Cannabinoid Receptor Interacting Protein (CRIP).  These studies will assess the potential for drugs to promote partnering with different G proteins and accessory proteins and thereby direct the selection of signal transduction pathways (e.g., via adenylyl cyclase inhibition, MAPK activation, Ca2+ regulation, and NO production). The importance to drug discovery is the promotion of therapeutically beneficial actions (e.g., analgesia) with diminished untoward side effects (e.g., sedation, cognitive dysfunction).

 

Dr. Howlett’s research investigates cannabinoid receptors, the G-protein coupled receptors that respond to a family of eicosanoid ligands in the body (e.g., anandamide, 2-arachidonoylglycerol), as well as to drugs (Δ9-tetrahydrocannabinol, CP-55940 and WIN55212-2).  The CB1 cannabinoid receptors are found abundantly in the nervous system, and less abundantly in many other organs.  The CB2 receptors are more readily characterized in the immune system.  Evidence exists for other subtypes.  We are interested in the mechanisms by which different drugs can activate G-proteins selectively to allow selection of therapeutically-relevant signal transduction pathways.

The working hypotheses begin with a model of sequential steps of ligand docking to the inactive receptor-GGDP complex; agonist-induced conformational change in the receptor and concomitant conformational change in the G protein resulting in it’s activation (GDP/GTP exchange); G or G regulation of effectors leading to signal transduction pathway activation. Our studies demonstrated that the G-protein selectivity that CB1 intracellular domains prefer. We further demonstrated that agonists of distinct chemical classes influence these different receptor-G protein interactions. 

We propose that chemically distinct CB1 ligands can change the conformation of the protein.  In addition, phosphorylation can define which signal transduction pathway(s) are activated.  We are working to define the types of G-proteins that interact with the CB1 receptor, and investigate the necessary receptor domains, and the influence of agonists and phosphorylation.

We are investigating the CB1 receptor intracellular surface structure that interfaces with G-proteins.  In these studies, we can assess modifications imposed by post-translational events such as phosphorylation.   We are also determining the potential for agonist-directed selection of specifici signal transduction pathways.  Among those down-stream pathways, we know that adenylyl cyclase inhibition, MAPK activation, Ca2+ regulation, and NO production, are only some of those intracellular events within this diversity of potential signal transduction pathway mechanisms.

The opportunity for agonist drug design to facilitate selective G-protein-mediated signal transduction pathways may allow stimulation of neuronal cells in brain regions that regulate beneficial effects such as pain relief and alleviation of spasticity and motor dysfunction of neurodegenerative diseases, while not affecting neurons in brain regions that promote untoward responses such as cognitive dysfunction and memory dysregulation.

 

Howlett, A.C., Breivogel, C.S., Childers, S.R., Deadwyler, S.A., Hampson, R.E. and Porrino, L.J. (2004) Cannabinoid physiology and pharmacology: 30 years of progress.  Neuropharmacology 47S1:345-358. Mukhopadhyay, S. and Howlett, A.C. (2005) Chemically distinct ligands promote differential CB1 cannabinoid receptor-Gi protein interactions. Mol. Pharmacol. 67: 2016-2024. Howlett, A.C., Mukhopadhyay, S. and Norford, D.C. (2006) Endocannabinoids and reactive nitrogen and oxygen species in neuropathologies. J. Neuroimmune Pharmacol. 1:305-316. Ward, G.R., Franklin, S.O., Dempsey, K.T., Clodfelter, D.E., Gerald, T.M., Krissinger, D.J., Patel, K.M., Vrana, K.E. and Howlett, A.C. (2007) Glucocorticoids plus opioids up-regulate genes that influence neuronal function. Cell Mol. Neurobiol. 27:651-660. Grace, C.R.R., Cowsik, S.M., Shim, J.-Y., Welsh, W.J., and Howlett, A.C. (2007) Conformation of a peptide mimetic of the fourth cytoplasmic loop of the CB1 cannabinoid receptor. J. Structural Biol. 159:359-368. Anavi-Goffer, S., Fleischer, D.,  Hurst, D. P., Lynch, D. L., Barnett-Norris, J., Shi, S., Lewis, D. L., Mukhopadhyay, S., Howlett, A.C., Reggio, P. H. and Abood, M. E. (2007)  Helix 8 Leu in the CB1 Cannabinoid Receptor contributes to selective coupling with G proteins. J. Biol. Chem. 282:25100-13. Niehaus, J.L., Liu, Y., Wallis, K.T., Egertova, M., Bhartur, S.G., Mukhopadhyay, S., Shi, S., He, H.,  Selley, D.E., Howlett, A.C., Elphick, M.R., and Lewis, D.L. (2007) CB1 cannabinoid receptor activity is modulated by the cannabinoid receptor interacting protein CRIP1a. Mol. Pharmacol. 72: 1557-1566. Jones, J.D., Carney, S.T., Vrana, K. E., Norford, D.C. and Howlett, A.C. (2008)  Cannabinoid receptor-mediated production of cyclic GMP and translocation of NO-sensitive guanylyl cyclase in neuronal cells. Neuropharmacology 54:23-30. Padgett, L.W., Howlett, A.C. and Shim, J.-Y. (2008) Binding mode prediction of conformationally restricted anandamide analogs within the CB1 receptor. J. Molec. Signaling 3:5.