Constitutive Activity of GPR40/FFA1

Stoddart, Leigh A.; Milligan, Graeme

doi:10.1016/b978-0-12-381298-8.00028-9

Cited by 8 publications

(3 citation statements)

References 45 publications

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“…The Flp-In T-REx cells provide an optimal experimental system to examine this, as they allow direct comparison of basal signaling within the same cell line, either with or without induced receptor expression. However, the assays used to describe the signaling properties of hFFA2-C4.57G/H6.55Q, including cAMP, Ca 2+ , pERK, and DMR, are not well suited to measuring ligand-independent constitutive activity; therefore, we extended our studies to measure incorporation of [ 35 S]GTPγS, an assay that has been widely used previously to study GPCR constitutive activity (28–29). Initially, we demonstrated that hFFA2 activity can be measured in this assay, as C3 (pEC 50 =4.16±0.11) but not compound 25 stimulated [ 35 S]GTPγS incorporation in membranes induced to express wild-type hFFA2 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs

et al. 2012

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When it is difficult to develop selective ligands within a family of related G-protein-coupled receptors (GPCRs), chemically engineered receptors activated solely by synthetic ligands (RASSLs) are useful alternatives for probing receptor function. In the present work, we explored whether a RASSL of the free fatty acid receptor 2 (FFA2) could be developed on the basis of pharmacological variation between species orthologs. For this, bovine FFA2 was characterized, revealing distinct ligand selectivity compared with human FFA2. Homology modeling and mutational analysis demonstrated a single mutation in human FFA2 of C4.57G resulted in a human FFA2 receptor with ligand selectivity similar to the bovine receptor. This was exploited to generate human FFA2-RASSL by the addition of a second mutation at a known orthosteric ligand interaction site, H6.55Q. The resulting FFA2-RASSL displayed a >100-fold loss of activity to endogenous ligands, while responding to the distinct ligand sorbic acid with pEC50 values for inhibition of cAMP, 5.83 ± 0.11; Ca2+ mobilization, 4.63 ± 0.05; ERK phosphorylation, 5.61 ± 0.06; and dynamic mass redistribution, 5.35 ± 0.06. This FFA2-RASSL will be useful in future studies on this receptor and demonstrates that exploitation of pharmacological variation between species orthologs is a powerful method to generate novel chemically engineered GPCRs.—Hudson, B. D., Christiansen, E., Tikhonova, I. G., Grundmann, M., Kostenis, E., Adams, D. R., Ulven, T., Milligan, G. Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs.

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Section: Resultsmentioning

confidence: 99%

Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs

et al. 2012

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“…9). In the absence of ALA a significantly higher level of [ 35 S]-GTPγS binding was seen in CHO-AEQ-FFA1 versus CHO-AEQ cell membranes which might be indicative for constitutive activity of the FFA1 receptor and/or the binding of endogenous free fatty acids, which were released from the cell during the membrane preparation and are able to bind the receptor [74,75]. This would also be consistent with the observation that a higher concentration of ALA is needed to inhibit [ 35 S]-GTPγS binding in CHO-AEQ-FFA1 as compared to CHO-AEQ cell membranes (Fig.…”

Section: Ala Inhibits [ 35 S]-gtpγs Binding In Cho-aeq Cell Membranesmentioning

confidence: 99%

Complex FFA1 receptor (in)dependent modulation of calcium signaling by free fatty acids

Katsouri

Vandervelpen²,

Gattor³

et al. 2022

Biochemical Pharmacology

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“…For example, a very recent study demonstrated that while FFA3 responded to SCFAs to induce sympathetic outflow after a meal, this receptor was antagonized by the ketone β-hydroxybutyrate, thought to be the HCA 2 endogenous agonist (Taggart et al, 2005), in times of starvation or in diabetes (Kimura et al, 2011). In another case, the apparent constitutive activity of FFA1 in [ 35 S]GTPγS activation assays was demonstrated to actually result from ligand-mediated receptor activation due to endogenous agonists released during membrane preparation (Stoddart et al, 2007; Stoddart and Milligan, 2010). It seems likely that further unexpected interactions at these receptors will be discovered in the future.…”

Section: Target Specificity/selectivitymentioning

confidence: 99%

Low Affinity GPCRs for Metabolic Intermediates: Challenges for Pharmacologists

Smith¹

2012

Front. Endocrin.

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The discovery that a number of metabolites and metabolic intermediates can act through G protein-coupled receptors has attracted great interest in the field and has led to new therapeutic targets for diseases such as hypertension, type 2 diabetes, inflammation, and metabolic syndrome. However, the low apparent affinity of these ligands for their cognate receptors poses a number of challenges for pharmacologists interested in investigating receptor structure, function or physiology. Furthermore, the endogenous ligands matched to their receptors have other, well established metabolic roles and thus selectivity is difficult to achieve. This review discusses some of the issues researchers face when working with these receptors and highlights the ways in which a number of these obstacles have been overcome.

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Constitutive Activity of GPR40/FFA1

Cited by 8 publications

References 45 publications

Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs

Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs

Complex FFA1 receptor (in)dependent modulation of calcium signaling by free fatty acids

Low Affinity GPCRs for Metabolic Intermediates: Challenges for Pharmacologists

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