Biochemical Pharmacology Core

Lee-Yuan Liu-Chen, Core Director ([click-for-email])

The Biochemical Pharmacology Core provides support of techniques that allow interrogation of receptors associated with drugs of abuse and their signaling mechanisms.

Specifically, the Core provides the following services:

• Radioligand binding to mu, delta and kappa opioid receptors (MOPR, DOPR, KOPR), the  nociceptin / orphanin FQ receptor (NOPR), as well as to the dopamine, sigma, cannabinoid (CB1 and CB2) and nicotinic receptors. Assays for other receptors or binding sites are developed when necessary.
• Assessment of G protein-coupled receptor (GPCR) activation: cAMP level, [³⁵S]GTPγS binding, p44/42 MAP kinases phosphorylation, receptor internalization, β-arrestin recruitment and G12/13 activation
• Determination of agonist bias at GPCRs: [³⁵S]GTPγS binding or cAMP and β-arrestin recruitment; agonist biases for CB1 and CB2 receptors using CRISPR cell lines with either individual G proteins deleted or arrestins deleted
• Receptor autoradiography of opioid and dopamine receptors in brain sections. Autoradiography for other receptors are developed when necessary.
• Autoradiography of opioid agonist-promoted [³⁵S]GTPγS in brain sections
• Immunoblotting of MOPR, KOPR and phosphorylated KOPR in cultured cells and in rodent brains
• Internalization of MOPR, DOPR and KOPR and CB1 in cultured cells and KOPR in mouse brains
• Providing purified MOPR, KOPR and phospho-KOPR antibodies to other researchers
• Training of personnel from other laboratories in the above techniques if the personnel in those laboratories desire to learn the techniques themselves

In recent years, it has become evident that biased agonists for GPCRs may preserve beneficial therapeutic effects with reduced side effects compared with balanced agonists. The Core has assays for G protein signaling, such as cAMP assay and [³⁵S]GTPγS binding, and has developed β-arrestin1 and β-arrestin2 recruitment assays. The Core has obtained CRISPR cell lines with either individual G proteins or arrestins deleted for investigation of CB1 and CB2 agonist biases. Thus, the Core is in a good position to determine agonist bias for opioid and CB receptors and potentially for other GPCRs.

It has been difficult to obtain selective antibodies for GPCRs. Dr. Liu-Chen’s lab has generated antiserum against a MOPR peptide, a KOPR peptide (371-380), a phospho-Thr363 (pT363) KOPR peptide, and a phospho-S368 (pS369) KOPR peptide.  Some of the affinity-purified antibodies showed high specificity in immunoblotting of endogenous receptors in the rat and mouse brain using knockout mice as negative controls. These antibodies are valuable tools for the opioid receptor field and are available for projects in the Core or can be shared with interested investigators for use in their own laboratories.

Dr. Liu-Chen’s laboratory has generated a mutant mouse line expressing a fusion protein of the KOPR with tdTomato, and shown that these mice allow visualization of agonist-induced KOPR internalization.