bret-based-g-protein-biosensors-for-measuring-g-protein-coupled

BRET-based G Protein Biosensors for Measuring G Protein-Coupled Receptor Activity in Live Cells

G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane receptors, playing a crucial role in cellular signaling by transducing ...

Philipps-University Marburg

Within the intestine, the human G protein-coupled receptor (GPCR) GPR35 is involved in oncogenic signaling, bacterial infections, and inflammatory bowel disease. GPR35 is known to be expressed as two distinct isoforms that differ only in the length of their extracellular N-termini by 31 amino acids, but detailed insights into their functional differences are lacking. Through gene expression analysis in immune and gastrointestinal cells, we show that these isoforms emerge from distinct promoter usage and alternative splicing. Additionally, we employed optical assays in living cells to thoroughly profile both GPR35 isoforms for constitutive and ligand-induced activation and signaling of 10 different heterotrimeric G proteins, ligand-induced arrestin recruitment, and receptor internalization. Our results reveal that the extended N-terminus of the long isoform limits G protein activation yet elevates receptor-β-arrestin interaction. To better understand the structural basis for this bias, we examined structural models of GPR35 and conducted experiments with mutants of both isoforms. We found that a proposed disulfide bridge between the N-terminus and extracellular loop 3, present in both isoforms, is crucial for constitutive G activation, while an additional cysteine contributed by the extended N-terminus of the long GPR35 isoform limits the extent of agonist-induced receptor-β-arrestin2 interaction. The pharmacological profiles and mechanistic insights of our study provide clues for the future design of isoform-specific GPR35 ligands that selectively modulate GPR35-transducer interactions and allow for mechanism-based therapies against, for example, inflammatory bowel disease or bacterial infections of the gastrointestinal system.

Isoforms of GPR35 have distinct extracellular N-termini that allosterically modify receptor-transducer coupling and mediate intracellular pathway bias.

In this study, we synthesized and evaluated new photoswitchable ligands for the beta-adrenergic receptors β-AR and β-AR, applying an azologization strategy to the first-generation beta-blocker propranolol. The resulting compounds (Opto-prop-1, -2, -3) have good photochemical properties with high levels of light-induced - isomerization (>94%) and good thermal stability (  > 10 days) of the resulting -isomer in an aqueous buffer. Upon illumination with 360-nm light to PSS  , large differences in binding affinities were observed for photoswitchable compounds at β-AR as well as β-AR. Notably, Opto-prop-2 (VUF17062) showed one of the largest optical shifts in binding affinities at the β-AR (587-fold, -active), as recorded so far for photoswitches of G protein-coupled receptors. We finally show the broad utility of Opto-prop-2 as a light-dependent competitive antagonist of the β-AR as shown with a conformational β-AR sensor, by the recruitment of downstream effector proteins and functional modulation of isolated adult rat cardiomyocytes.

Optical control of the β-adrenergic receptor with opto-prop-2: A -active azobenzene analog of propranolol.

A large portion of the human GPCRome is still in the dark and understudied, consisting even of entire subfamilies of GPCRs such as odorant receptors, class A and C orphans, adhesion GPCRs, Frizzleds and taste receptors. However, it is undeniable that these GPCRs bring an untapped therapeutic potential that should be explored further. Open questions on these GPCRs span diverse topics such as deorphanisation, the development of tool compounds and tools for studying these GPCRs, as well as understanding basic signalling mechanisms. This review gives an overview of the current state of knowledge for each of the diverse subfamilies of understudied receptors regarding their physiological relevance, molecular mechanisms, endogenous ligands and pharmacological tools. Furthermore, it identifies some of the largest knowledge gaps that should be addressed in the foreseeable future and lists some general strategies that might be helpful in this process. LINKED ARTICLES: This article is part of a themed issue Complexity of GPCR Modulation and Signaling (ERNST). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v182.14/issuetoc.

The dark sides of the GPCR tree - research progress on understudied GPCRs.

G protein-coupled receptors (GPCRs) are a large family of cell surface receptors that play a critical role in nervous system function by transmitting signals between cells and their environment. They are involved in many, if not all, nervous system processes, and their dysfunction has been linked to various neurological disorders representing important drug targets. This overview emphasises the GPCRs of the nervous system, which are the research focus of the members of ERNEST COST action (CA18133) working group 'Biological roles of signal transduction'. First, the (patho)physiological role of the nervous system GPCRs in the modulation of synapse function is discussed. We then debate the (patho)physiology and pharmacology of opioid, acetylcholine, chemokine, melatonin and adhesion GPCRs in the nervous system. Finally, we address the orphan GPCRs, their implication in the nervous system function and disease, and the challenges that need to be addressed to deorphanize them. LINKED ARTICLES: This article is part of a themed issue Complexity of GPCR Modulation and Signaling (ERNST). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v182.14/issuetoc.

ERNEST COST action overview on the (patho)physiology of GPCRs and orphan GPCRs in the nervous system.

A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cβ (PLCβ) isozymes to increase cytosolic Ca in living cells is exclusive to Gq- and Gi-sensitive G protein-coupled receptors (GPCRs). Here we extend this central tenet and show that Gs-GPCRs also partake in inositol-lipid signaling and thereby increase cytosolic Ca. By combining CRISPR/Cas9 genome editing to delete Gα, the adenylyl cyclase isoforms 3 and 6, or the PLCβ1-4 isozymes, with pharmacological and genetic inhibition of Gq and G11, we pin down Gs-derived Gβγ as driver of a PLCβ2/3-mediated cytosolic Ca release module. This module does not require but crosstalks with Gα-dependent cAMP, demands Gα to release PLCβ3 autoinhibition, but becomes Gq-independent with mutational disruption of the PLCβ3 autoinhibited state. Our findings uncover the key steps of a previously unappreciated mechanism utilized by mammalian cells to finetune their calcium signaling regulation through Gs-GPCRs.

Article	Year
Isoforms of GPR35 have distinct extracellular N-termini that allosterically modify receptor-transducer coupling and mediate intracellular pathway bias. The Journal of biological chemistry\| PubMed ID: 35933013	2022
Optical control of the β-adrenergic receptor with opto-prop-2: A -active azobenzene analog of propranolol. iScience\| PubMed ID: 36060054	2022
The dark sides of the GPCR tree - research progress on understudied GPCRs. British journal of pharmacology\| PubMed ID: 38339984	2025
ERNEST COST action overview on the (patho)physiology of GPCRs and orphan GPCRs in the nervous system. British journal of pharmacology\| PubMed ID: 38825750	2025
A molecular mechanism to diversify Ca signaling downstream of Gs protein-coupled receptors. Nature communications\| PubMed ID: 39227390	2024

Hannes Schihada

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