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A small fragment (SmBiT) was N-terminally fused to a C68-mutated Gγ2 subunit with a 15-amino acid flexible linker. HEK293T cells were seeded in a six-well plate and allowed to grow to 80% confluence before transfection. For measuring Gs signaling, a plasmid mixture consisting of 200 ng LgBiT-inserted Gαs subunit, 1000 ng Gβ1, 1000 ng C68S-mutant SmBiT-fused Gγ2 (C68S), 400 ng test GPCR in 200 μL of Opti-MEM (Gibco) was transiently transfected with PEI. After 1-day transfection, transfected cells were plated onto a 96-well plate treated by cell adherent reagent (Applygen).
Other constructs including the full-length and various site-directed mutated human CCK1R and CCK2R were cloned into the pcDNA3.1 vector for cAMP accumulation and NanoBiT–G protein dissociation assay. The intestinal hormone and neuromodulator cholecystokinin (CCK) receptors CCK1R and CCK2R act as a signaling hub in brain–gut axis, mediating digestion, emotion, and memory regulation. CCK receptors exhibit distinct preferences for ligands in different posttranslational modification (PTM) states. They exist in multiple molecular forms, sharing the same evolutionarily conserved pentapeptide motif, which comprises the minimal sequence required for biological activity and receptor activation (Fig. 1a).
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- The processing, however, of cardiac proCCK is unique, as the result is a long triple-sulfated and N-terminally truncated fragment 25–94 with only trace amounts of the conventionally amidated and sulfated CCK peptides (113).
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Adrenal medullary cells produce small amounts of CCK, although amidated and with a low degree of sulfation (98). The picture has varied (12) due to species differences and because the molecular pattern along the gut varies (59, 60). In man, CCK-33 predominates in plasma, but CCK-58, -22, and -8 are also present (11, 61). Moreover, cerebral CCK neurons are more abundant than neurons of other neuropeptides (42, 45, 46). While most peptidergic neurons occur in subcortical regions, CCK is expressed in the highest concentrations in neocortical neurons (39, 42, 47). The perikarya of the cortical CCK nerves are distributed in layers II–VI, with the highest frequency in layers II and III (42, 48).
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Participated in manuscript preparation; H.Z., L.-N.C., J.Q., D.-D.S., and C.M. Performed specimen screening, cryo-EM grid preparation, data collection; H.Z. Processed the cryo-EM data, modeled, and refined the structures from cryo-EM density maps; H.Z. Performed structural analysis and figure preparation; H.Z., Y.D., Y.-Y.L., L.-N.C., and S.-Y.J.
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