A, Diagram of the co- and posttranslational modifications of proCCK. Activation of the CCK amidation site (FGRR, sequence 83–86 of proCCK) occurs via a series of carboxyterminal cleavages and modifications. An early modification is tyrosyl O-sulfation by sulfotransferases of Y₇₇, Y₉₁, and Y₉₃. In addition to ensuring binding to the CCK-A receptor, tyrosyl sulfation also increases endoproteolytic cleavage (39). Endoproteolytic cleavage by a PC (PC1/3, PC2, PC5/6, or other PCs) produces the carboxypeptidase E substrate. Carboxypeptidase E then acts to remove the C-terminal arginyl residue yielding glycine-extended CCK, which by peptidylglycine α-amidating monooxygenase (PAM) results in the production of bioactive CCK (F-NH₂). Concomitant N-terminal cleavages by PCs produce bioactive CCKs of varying length. In combination with in vitro endoprotease treatment (trypsinization), a library of sequence-specific antibodies was used to measure bioactive CCK and precursor peptides, as described in the text. Although the text enlists only CCK-58, -33, -22, and -8 as bioactive CCKs, small amounts of CCK-83 and -5 may also be synthesized in the brain and gut (12). B, Binding sites of antibodies (AB) for the study of endoproteolytic proCCK processing: AB 92128 binds proCCK sequence 76–83 in its derivatized form, i.e. Tyr₇₆ is O sulfated (marked with an asterisk) and Phe₈₃ carboxyamidated. This structure constitutes the C terminus of the bioactive cholecystokinins (CCK-83,-58,-33,-22, and -8). AB 2609 binds the same sequence as AB 92128 but is independent of tyrosyl O-sulfation. Consequently, it binds both sulfated and nonsulfated CCK-peptides. AB 3208 binds proCCK sequence 76–84, i.e. the glycine-extended forms of CCK that constitute the immediate precursors of the bioactive carboxyamidated CCKs. AB 89009 binds proCCK sequence 62–68, corresponding to the N terminus of human CCK-22. Consequently, AB 89009 binds both CCK-22 and C-terminal extended forms of CCK-22 such as glycine-extended CCK-22. Further details of the epitope specificity of the antibodies are given elsewhere (8,25,26,27).

Gel chromatography on Sephadex G-50 superfine columns of neutral water extracts from the small intestine of PC1/3 knockout mice (KO; left panels) and from corresponding wild-type mice (WT; right panels). The chromatographic elutions of carboxyamidated and tyrosylsulfated CCKs were monitored by a RIA using the CCK-specific antibody no. 92128 (upper panels), whereas the elutions of proCCK fragments were monitored after trypsin-CPB treatment by a RIA using antibody no. 3208 specific for glycine-extended CCK (lower panels). Extracts from four different KO and WT mice small intestines, respectively, were subjected to chromatography. Those shown are characteristic of each group. The peptide concentrations in acetic acid extracts were too low for chromatography.

Gel chromatography on Sephadex G-50 superfine columns of neutral water extracts from the whole brain of PC1/3 knockout mice (KO; left panels) and from corresponding wild-type mice (WT; right panels). The chromatographic elutions of carboxyamidated and tyrosylsulfated CCKs were monitored by a RIA using the CCK-specific antibody no. 92128 (upper panels), whereas the elutions of proCCK fragments were monitored after trypsin-CPB treatment by a RIA using antibody no. 3208 specific for glycine-extended CCK (lower panels). Extracts from four different KO and WT mice brains, respectively, were subjected to chromatography. Those shown are characteristic of each group. The peptide concentrations in acetic acid extracts were too low for chromatography. Although extracts of whole brain were used, it should be noted that nearly all CCK in the mammalian central nervous system is expressed in the cerebrum (3,4,12).

Gel chromatography on Sephadex G-50 superfine columns of a neutral water extract from SK-N-MC cells that express the CCK gene. The chromatographic fractions were monitored by RIAs using antibody (AB) no. 3208 specific for glycine-extended CCK and confirmed with antibody no. 89009 specific for the N terminus of human CCK-22 (upper panel) or after trypsin-CPB treatment using the same RIAs (lower panel).

Immunhistochemical visualization of CCK (green) and PC1/3 (red) (A–C) in I cells of the mouse small intestine. Note the complete colocalization of PC1/3 and CCK in these cells (A–C). In contrast, CCK (green) and PC2 were found in different cells of the of the small intestine (D–F). Scale bars, 50 μm (A-C); 25 μm (D–F).

Immunohistochemical visualization of CCK (red) and PC2 (green) in the mouse cerebral cortex (A–F) and CCK (red) and PC1/3 (green) in the hypothalamic supraoptic nucleus (G–I). Note that all CCK-expressing neurons also express PC2 (arrows in A–C), whereas no colocalization were found between CCK and PC1/3 (G–I). Scale bars, 50 μm (A-C); 20 μm (D–F).

Small intestinal colocalization of PC5/6 and CCK is shown in two different small intestinal samples. CCK-expressing cells were identified by immunohistochemistry (IHC) (B and D), whereas PC5/6-expressing cells were identified by in situ hybridization (A and C). The CCK-expressing cells that also express PC5/6 are indicated by an arrow with one arrowhead. Endocrine cells that express PC5/6 but not CCK are indicated by an arrow with two arrowheads. Lastly, a single cell that expresses CCK but not PC5/6 was also found and is indicated by an arrow with three arrowheads.