The 5-hydroxytryptamine (serotonin) receptor type 7 (5-HT7R) is a G protein-coupled receptor present primarily in the nervous system and gastrointestinal tract, where it regulates mood, cognition, digestion, and vasoconstriction. 5-HT7R has previously been shown to bind to its cognate stimulatory Gs protein in the inactive state. This phenomenon, termed "inverse coupling," is thought to counteract the atypically high intrinsic activity of 5-HT7R. However, it is not clear how active and inactive 5-HT7 receptors affect the mobility of the Gs protein in the plasma membrane. Here, we used single-molecule imaging of the Gs protein and 5-HT7R to evaluate Gs mobility in the membrane in the presence of 5-HT7R and its mutants. We show that expression of 5-HT7R dramatically reduces the diffusion rate of Gs. Expression of the constitutively active mutant 5-HT7R (L173A) is less effective at slowing Gs diffusion presumably due to the reduced ability to form long-lasting inactive complexes. An inactive 5-HT7R (N380K) mutant slows down Gs to the same extent as the wild-type receptor. We conclude that inactive 5-HT7R profoundly affects Gs mobility, which could lead to Gs redistribution in the plasma membrane and alter its availability to other G protein-coupled receptors and effectors.

Department of Molecular Biology and Genetics Faculty of Science University of South Bohemia CZ 37005 České Budějovice Czech Republic

Department of Pharmacology and Toxicology Augusta University Augusta GA 30912

Laboratory of Microscopy and Histology Institute of Entomology Biology Centre of the Czech Academy of Sciences CZ 37005 České Budějovice Czech Republic

Laboratory of Structural Biology and Bioinformatics Institute of Microbiology of the Czech Academy of Sciences CZ 37333 Nové Hrady Czech Republic

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