GPCR aggregation on the cell surface and internalization via clathrin-coated pits to intracellular compartments (Figure 1) is a universal mechanism for modulating and tuning of the response to the agonist1,2. In many GPCRs, engagement with β−arrestins precedes internalization and initiates further signaling cascades through ERK and other kinases. Because the receptor is no longer available for binding, internalization leads to “desensitization,” a decrease in receptor response even in the presence of elevated concentrations of the agonist. Although the majority of GPCRs are internalized through β−arrestin-mediated pathways, some can be internalized via β−arrestin-independent mechanisms. Internalization is therefore a key mechanism in modulating and directing GPCR response as well as an important consideration in the design of drugs which target this class.
Background To provide tools for GPCR screening and analysis, Multispan has developed stable mammalian cell lines expressing more than 200 GPCRs3 that are suitable for high throughput cell-based screening assays. Since GPCRs can couple to different G proteins and activate separate downstream signaling pathways (Figure 1,4) we have developed a comprehensive panel of functional assays to interrogate different receptor signaling pathways. Assay readouts include calcium, cAMP, β-arrestin, pGRK2, IP-1, pERK, pNFκB, radioligand binding, GTPγS, chemotaxis, cytokine secretion, insulin secretion, cell proliferation and luciferase reporters. Used in high throughput screens (HTS), these assays have enabled the identification of compounds that selectively activate a subset of these pathways for specific receptors.
However, these downstream signaling based HTS assays fail to measure receptor internalization through endocytosis and recycling to the cell surface over time (Figure 1). This signifies as a universal feature of GPCRs after its activation regardless of its ligands being known.
To circumvent this shortcoming, we have developed a generalized cell-based GPCR screening methodology for measurement of GPCR receptor internalization in response to ligand. Our assay quantifies the cell surface expression of the GPCR following exposure to the ligand. The assay is performed that can be used to screen the effect of compounds on receptor internalization in high throughput. Furthermore, it also permits screening of receptors independent of any downstream signaling.
Methodology The assay measures the expression of epitope-tagged GPCRs at the cell-surface using flow cytometry in 96-well plate format. The example in Figure 2 demonstrates measurement of the internalization of the CCK1 receptor in response to CCK treatment. Surface expression of CCK1 is decreased in both a dose- and time-dependent manner (panel A and B, and its pharmacological profiles closely correlate to those obtained by second messenger measurements (not shown). Furthermore, the agonist-induced effects can be reversed by receptor-specific antagonists (Figure 2 Panel C).
Figure 2 CCK1 CHO-K1 cells were treated with agonist CCK (26-33) for various times, stained with an anti-FLAG antibody and surface expression measured by FACS. Percentage of cell surface receptor was calculated from geometric mean of fluorescence intensities. In Panel C, treatment with the antagonist Lorglumide suppresses the effect of the agonist on receptor internalization
In another example, shown in Figure 3, the internalization assay was used as an important complement to other GPCR signaling assays in the analysis of signaling from the mu- (MOR) and kappa (KOR) opioid receptors. Furthermore, Pertussis toxin (PTX) impairs G protein heterotrimer interaction with receptors, thereby blocking Gαi/Gαo receptor coupling in both MOR and KOR pERK and cAMP assays, while having no effect on MOR internalization and only partially blocking KOR internalization.
Figure 3 Cells stably over-expressing MOR/KOR were treated with PTX or vehicle overnight prior to dose-dependent stimulation with control agonist and analyzed in pERK, Gαi -mediated cAMP, or internalization assays.
Applications These results suggest that measuring GPCR internalization using a live cell flow cytometry (FACS) is a simple cell-based approach to measure receptor response without bias toward any signaling pathway or knowledge of cellular mechanisms of orphan receptors. Use of a single well-characterized clonal cell line (see below) for a comprehensive assay panel avoids the requirement to maintain and compare results across assay-specific lines used in fluorescent energy transfer or enzyme fragment complementation assays. The live cell flow cytometry internalization assays can be readily adapted for HTS and has several important applications.
a) Hit compound characterization. There is broad interest in the development of improved pharmaceuticals that target key GPCR families5. Characterization of the kinetics and dose-dependence of the GPCR response is an important data element in triaging and prioritizing hit compound families. For example, screens of the Dopamine D1 receptor (D1R) have identified partially selective D1R agonists which do not promote receptor internalization8. It provides insight into unique biological functions of novel compounds to guide further medicinal chemistry. Moreover, this flow based assay to characterize drug effects on receptor internalization and recycling could also open new avenues to address receptor desensitization and drug tolerance for next generation opioids6 and beta blockers7.
b) Parallel primary screen. When used as a complementary primary screen, the internalization assay may identify hits that may be missed in primary screens that use a specific cell signaling readout of receptor activation.
c) Screens for orphan receptor agonists or surrogate ligands. For receptors for which the native agonist is unknown, the receptor internalization assay can be used as a primary screen to identify potential synthetic surrogate agonists that promote internalization. Synthetic agonists may offer important biological insights into the structure of the native agonist itself, provide useful probes of receptor function and serve as positive agonists for use in antagonist screens.
About the MULTISCREEN™ 231-GPCR Panel3 All receptor in Multispan’s flagship panel include the epitope-tag required for the MULTISCREEN™ Receptor Internalization Assay. Each GPCR target is expressed in a single, stable clonal carefully selected and rigorously characterized to ensure proper pharmacology with the receptor’s natural agonist. The expansive over 500 MULTISCREEN™ stable cell line clones can help jump start your discovery and understanding the MOA of your GPCR target, hit compound or IND-ready lead.
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3.Multispan, Inc | Scientific Insight: 231-GPCR Cell-Based Assay Panel (multispaninc.com)
4.Smith JS, Lefkowitz RJ, Rajagopal S. Biased signalling: from simple switches to allosteric microprocessors. Nat Rev Drug Discov. 2018;17(4):243-260.PMID 29302067
5.Multispan, Inc | Cell-Based Assay Development for GPCRs and Beyond
6.Williams JT, Ingram SL, Henderson G, Chavkin C, von Zastrow M, Schulz S, et al. Regulation of μ-opioid receptors: desensitization, phosphorylation, internalization, and tolerance. Pharmacol Rev. 2013 Jan;65(1):223–54PMID 23321159
7.Abosamak NER, Shahin MH. Beta 2 Receptor Agonists/Antagonists. [Updated 2022 Jul 4]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-.Available from: https://www.ncbi.nlm.nih.gov/books/NBK559069/
8.Conroy JL, Free RB, Sibley DR. Identification of G protein-biased agonists that fail to recruit β-arrestin or promote internalization of theD1 dopamine receptor. ACS Chem Neurosci. 2015;6(4):681-692PMID 25660762