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  • After 1-day transfection, transfected cells were plated onto a 96-well plate treated by cell adherent reagent (Applygen).

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Of particular importance is the tyrosyl residue in position seven as counted from the C-terminus (Figure 1). The CCK2 receptor binds sulfated and unsulfated ligands equally well, whereas the CCK1 receptor is exclusive and requires Y-sulfation of the ligand. In detail, both CCK1R and CCK2R could couple to Gq, whereas only CCK1R exhibits the Gs-coupling capability. Our CCK1R–Gs and CCK2R–Gq structures could provide a unique opportunity to understand the molecular basis for G protein-coupling specificity, at least for CCKRs.

Therefore, we attribute the fact that CCK2R recognizes non-sulfated and sulfated CCK-8 equally well to the lack of polar interactions between the endogenous ligands and CCK2R. CCK1R was co-expressed with human Gs protein in Sf9 insect cells to form the CCK1R–Gs complexes and activated by CCK-8 and by CCK1R-selective agonist SR146131, respectively. All complexes were purified to homogeneity for single-particle cryo-EM analysis (Supplementary Fig. S2). G protein activation was measured by a NanoBiT–G protein dissociation assay in which GPCR-induced G protein dissociation is monitored by a NanoBiT system. A large fragment (LgBiT) of the NanoBiT luciferase was inserted into the helical domain (between the αA and the αB helices) of a Gα subunit with 15-amino acid flexible linkers.

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Accordingly, adult cardiomyocytes in mice, rats, and pigs contain substantial amounts of proCCK protein (113). 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). The tissue concentration of the long proCCK fragment is higher in atrial than ventricular myocytes. The long proCCK fragment is released to plasma and may find use as a marker of the risk of mortality in heart failure patients (113). Cholecystokinin immunoreactivity has consistently been found to be expressed in human and rat mononuclear cells in blood (106, 107).

Only one CCK mRNA molecule has been found, and the CCK peptides are thus fragments of the same proCCK protein. The concentrations of CCK mRNA in cerebrocortical tissue are similar to that of the duodenal mucosa (34), and in the brain, there is a rapid synthesis of CCK peptides (35). The relatively high-resolution density maps of the four complexes allowed confident modeling of the CCK-8 and SR molecules, the N-terminal 8 residues of gastrin-17, most portions of the two receptors, and their corresponding G proteins. Spermatogenic cells express transiently the CCK gene in most mammals (99, 100). Interestingly, the CCK peptides in mature spermatozoes are concentrated in the acrosomal granule, which opens the possibility that CCK may play a role in fertilization due to the acrosomal reaction (100). The acrosomal expression is species-specific, as human spermatozoes in addition to CCK also express its homolog, gastrin (101).

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All known biological effects of CCK peptides reside in the conserved C-terminal heptapeptide sequence (Figure 1). Modification of this sequence grossly reduces or abolishes receptor binding and biological effects (13–15). The N-terminal extensions of the common C-terminus increase the biological potency and the specificity for receptor binding.

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