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We compared the CCK1R–Gs complex with other GPCR–Gs protein complexes. In each panel (Supplementary Fig. S5a, f), the complexes were aligned by the receptor, thus showing differences in the orientation of the Gs protein relative to the receptor. The αN orientation of CCK1R–Gs is closest to that of A2AR–Gs and β2AR–Gs, whereas the conformation of the α5 helix shows marked differences when compared to that in other GPCR–Gs complexes. Owning to the unique transducer–pocket conformation, the extreme C-terminus of Gs-α5 helix unexpectedly inserts into the cleft between TM6 and the TM7–H8 hinge (Supplementary Fig. S5b).
Cholecystokinin—From Local Gut Hormone to Ubiquitous Messenger
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- Note the unique disulfated sequence, which might suggest that cionin may resemble a common ancestor of CCK and gastrin.
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Protein concentration was determined by absorbance at 280 nm using a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific). The sample-coated grids were blotted before plunge-freezing into liquid ethane and stored in liquid nitrogen for data collection. The microscope was operated at 300 kV accelerating voltage, at a nominal magnification of 29,000 in counting mode, corresponding to a pixel size of 1.014 Å. A Electrostatic potential map of the binding pocket in CCK1R for the sulfated tyrosine of CCK-8. B The effect of N98A, R197A, or R197M mutation in CCK1R on CCK-8-induced cAMP accumulation. C Electrostatic potential map of the binding pocket in CCK2R for the sulfated tyrosine of CCK-8.
The particles were extracted and subjected to 3D classification on the complex, and the best-resolved class was selected. The resulting set of 299,092 particles was subjected to CTF refinement, Bayesian polishing and 3D auto refinement using the pixel size of 1.014 Å. The final map has an indicated global resolution of 3.2 Å at a Fourier shell correlation (FSC) of 0.143.
The assays have confirmed that protein- and fat-rich food is the most important stimulus (11, 54). Of the constituents, protein and l -amino acids as well as digested fat cause significant CCK release (54, 55). Carbohydrates only release small amounts of CCK (54), but hydrochloric acid also stimulates release (55). As described earlier (9), “the exomal unit of the CCK gene is seven kilobases interrupted by two introns (23). Whereas the function of the E-box and the GC-rich region is not fully clarified (26, 27), the combined CRE/TRE sequence plays an important role in the regulation of CCK transcription.