A complex evaluation of agonist bias at G-protein coupled receptors at the level of G-protein classes and isoforms including non-preferential ones is essential for advanced agonist screening and drug development. Molecular crosstalk in downstream signaling and a lack of sufficiently sensitive and selective methods to study direct coupling with G-protein of interest complicates this analysis. We performed binding and functional analysis of 11 structurally different agonists on prepared fusion proteins of individual subtypes of muscarinic receptors and non-canonical promiscuous α-subunit of G16 protein to study agonist bias. We have demonstrated that fusion of muscarinic receptors with Gα16 limits access of other competitive Gα subunits to the receptor, and thus enables us to study activation of Gα16 mediated pathway more specifically. Our data demonstrated agonist-specific activation of G16 pathway among individual subtypes of muscarinic receptors and revealed signaling bias of oxotremorine towards Gα16 pathway at the M2 receptor and at the same time impaired Gα16 signaling of iperoxo at M5 receptors. Our data have shown that fusion proteins of muscarinic receptors with α-subunit of G-proteins can serve as a suitable tool for studying agonist bias, especially at non-preferential pathways.

• Klíčová slova
• fusion proteins, muscarinic receptors, non-canonical signaling, signaling bias,
• MeSH
• AMP cyklický metabolismus   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• inhibiční koncentrace 50 MeSH
• isoxazoly chemie   MeSH
• křečci praví MeSH
• kvartérní amoniové sloučeniny chemie   MeSH
• lidé MeSH
• molekulární konformace MeSH
• oxotremorin chemie   MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 metabolismus   MeSH
• receptory muskarinové metabolismus   MeSH
• rekombinantní fúzní proteiny chemie   MeSH
• signální transdukce * MeSH
• simulace molekulární dynamiky MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• AMP cyklický MeSH
• G protein alpha 16 MeSH Prohlížeč
• iperoxo MeSH Prohlížeč
• isoxazoly MeSH
• kvartérní amoniové sloučeniny MeSH
• oxotremorin MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 MeSH
• receptory muskarinové MeSH
• rekombinantní fúzní proteiny MeSH

BACKGROUND AND PURPOSE: More than 30% of currently marketed medications act via GPCRs. Thus, GPCRs represent one of the most important pharmacotherapeutic targets. In contrast to traditional agonists activating multiple signalling pathways, agonists activating a single signalling pathway represent a new generation of drugs with increased specificity and fewer adverse effects. EXPERIMENTAL APPROACH: We have synthesized novel agonists of muscarinic ACh receptors and tested their binding and function (on levels of cAMP and inositol phosphates) in CHO cells expressing individual subtypes of muscarinic receptors, primary cultures of rat aortic smooth muscle cells and suspensions of digested native tissues from rats. Binding of the novel compounds to M2 receptors was modelled in silico. KEY RESULTS: Two of the tested new compounds (1-(thiophen-2-ylmethyl)-3,6-dihydro-2H-pyridinium and 1-methyl-1-(thiophen-2-ylmethyl)-3,6-dihydro-2H-pyridinium) only inhibited cAMP synthesis in CHO cells, primary cultures, and native tissues, with selectivity for M2 muscarinic receptors and displaying bias towards the Gi signalling pathway at all subtypes of muscarinic receptors. Molecular modelling revealed interactions with the orthosteric binding site in a way specific for a given agonist followed by agonist-specific changes in the conformation of the receptor. CONCLUSIONS AND IMPLICATIONS: The identified compounds may serve as lead structures in the search for novel non-steroidal and non-opioid analgesics acting via M2 and M4 muscarinic receptors with reduced side effects associated with activation of the phospholipase C signalling pathway.

• MeSH
• agonisté muskarinových receptorů * farmakologie   MeSH
• antagonisté muskarinových receptorů farmakologie   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• křečci praví MeSH
• krysa rodu Rattus MeSH
• receptor muskarinový M2 MeSH
• receptory muskarinové * MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• agonisté muskarinových receptorů * MeSH
• antagonisté muskarinových receptorů MeSH
• receptor muskarinový M2 MeSH
• receptory muskarinové * MeSH

We have found earlier that changes in membrane cholesterol content have distinct impact on signaling via the M1, M2, or M3 receptors expressed in CHO cells (CHO-M1 through CHO-M3). Now we investigated whether gradual changes in membrane cholesterol exerts differential effects on coupling of the M1 and M3 muscarinic receptors to preferential signaling pathways through Gq/11 and non-preferential Gs G-proteins signaling. Changes in membrane cholesterol resulted in only marginal alterations of antagonist and agonist affinity of the M1 and M3 receptors, and did not influence precoupling of either subtype. Changes in membrane cholesterol did not influence parameters of carbachol-stimulated GTP-γ(35)S binding in CHO-M1 membranes while reduction as well as augmentation of membrane cholesterol lowered the efficacy but increased the potency of carbachol in CHO-M3 membranes. Gradual increase or decrease in membrane cholesterol concentration dependently attenuated agonist-induced inositolphosphates release while only cholesterol depletion increased basal values in both cell lines. Similarly, membrane cholesterol manipulation modified basal and agonist-stimulated cAMP synthesis via Gs in the same way in both cell lines. These results demonstrate that changes in membrane cholesterol concentration differentially impact preferential and non-preferential M1 and M3 receptor signaling. They point to the activated G-protein/effector protein interaction as the main site of action in alterations of M1 receptor-mediated stimulation of second messenger pathways. On the other hand, modifications in agonist-stimulated GTP-γ(35)S binding in CHO-M3 membranes indicate that in this case changes in ligand-activated receptor/G-protein interaction may also play a role.

• Klíčová slova
• Agonist binding, Cholesterol, G-Proteins, Muscarinic receptors, Signal transduction, cAMP synthesis,
• MeSH
• CHO buňky MeSH
• cholesterol metabolismus   MeSH
• Cricetulus MeSH
• karbachol farmakologie   MeSH
• lidé MeSH
• proteiny vázající GTP metabolismus   MeSH
• receptor muskarinový M1 účinky léků   metabolismus   MeSH
• receptor muskarinový M3 účinky léků   metabolismus   MeSH
• signální transdukce MeSH
• systémy druhého messengeru fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholesterol MeSH
• karbachol MeSH
• proteiny vázající GTP MeSH
• receptor muskarinový M1 MeSH
• receptor muskarinový M3 MeSH