Roth Lab Research

The ultimate goal of our studies is to discover novel ways to treat human disease using GPCR-based therapeutics.

G-protein coupled receptors (GPCRs), which are also known as 7-TM or heptahelical receptors, represent a large and diverse family of proteins encompassing more than 1% of the genome. As shown in Figs 1 and 2 (from Kroeze et al J Cell Sci. 2003 and Armbruster et al JBC 2004, respectively) GPCRs have 7-TM domains and couple to multiple intracellular signaling pathways. Currently, more than 50% of approved medications target GPCRs (Roth et al)

Figure 1: G-Protein Coupled Receptors at a Glance; Kroeze et al

Figure 2: Functional Assays Based on Diverse Signaling and Regulation of GPCRs

G protein activation. Agonist induced GPCRs activation results in the exchange of GDP with GTP within Gα and can be monitored using non-hydrolyzable 35S-GTP[γS]. The intrinsic GTPase activity of Gα (release of 32P-Pi from [γ-32P]-GTP), which is enhanced by RGS proteins, has also been assayed.

Second messenger production by G protein activated effectors. The modulation of G protein effectors (e.g., Gαs stimulation and Gαi/o inhibition of AC stimulated cAMP production or activation of PLCβ by Gαq/11) are well established and the principal assays for examining GPCR activation. Fluorescent measurement of intracellular Ca2+ release resulting from PLCβ driven IP3 production is a convenient functional assay platform. Similarly, other G protein activated effectors, such as ion channels, may be monitored by fluorescent dyes that bind to ions and/or respond to changes in membrane potential or pH.

GPCR desensitization and receptor membrane expression. Following agonist activation, GPCRs are desensitized via phosphorylation and internalization into endosomes and are either recycled back to the cell surface or degraded in lysosomes. HT assays have been designed to monitor transitions in cellular localization of proteins involved in desensitization (e.g., transient redistribution of β-arrestins from the cytosol to the membrane following agonist treatment, (Ferguson et al) or enhanced cell surface expression of wildtype and constitutively active GPCRs by inverse agonists (Menzaghi et al, Parnot et al)).

Transcriptional activation following GPCR stimulation. A number of reporter assays have been developed to monitor the transcriptional activation of reporter genes and/or cell proliferation in response to GPCR-mediated activation of MAPK pathways (Weiner et al, Burstein et al).

Ligand-induced GPCR dimerization. Co-expression of GPCRs differentially tagged with 19 fluorescent markers has been used in combination with resonance energy transfer methods, such as fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET), to identify agonists based on spectrum shifts resulting from ligand-induced receptor dimerization and/or G protein dissociation (Janetopoulos et al). AC, adenylate cyclase; GIRK, G-protein-activated inwardly rectifying K+ channel; VDCC, voltage-dependent Ca2+ channel; PLCβ, phospholipase Cβ GRK, G-protein-coupled receptor kinase; βARR, β-arrestin; PIP2, phosphatidylinositol; DAG, diacylglycerol; IP3, inositol trisphosphate; RAF1, MEK1, ERK1/2 mitogen-activated protein kinases; and RGS, regulator of G protein signaling.