Effects of cholecystokinin-58 on type 1 cholecystokinin receptor function and regulation

Abstract

Cholecystokinin, like many peptide hormones, is present as multiple molecular forms. CCK-58 has been identified as the dominant form in the circulation, whereas most of the studies of CCK-receptor interactions have been performed with CCK-8. Despite both sharing the pharmacophoric region of CCK, representing its carboxy terminal heptapeptide amide, studies in vivo have demonstrated biological diversity of action of the two peptides, with CCK-58, but not CCK-8, stimulating pancreatic fluid secretion and lengthening the interval between meals. Here, we have directly studied the ability of these two CCK peptides to bind to the type 1 CCK receptor and to stimulate it to elicit an intracellular calcium response. The calcium response relative to receptor occupation was identical for CCK-58 and CCK-8, with the longer peptide binding with approximately fivefold lower affinity. We also examined the ability of the two peptides to elicit receptor internalization using morphological techniques and to disrupt the constitutive oligomerization of the CCK receptor using receptor bioluminescence resonance energy transfer. Here, both full agonist peptides had similar effects on these regulatory processes. These data suggest that both molecular forms of CCK act at the CCK1 receptor quite similarly and elicit similar regulatory processes for that receptor, suggesting that the differences in biological activity observed in vivo most likely reflect differences in the clearance and/or metabolism of these long and short forms of CCK peptides.

Fig. 1.

CCK-58 and CCK-8 receptor binding and biological activity. Shown are competition-binding curves (A) and stimulated intracellular calcium response (Δ[Calcium]_i) curves (B) to CCK-8 and CCK-58 in rat and human CCK₁ receptor-bearing cells. All values represent the means ± SE of data from at least 3 independent experiments (for binding) and duplicate determinations (for calcium). rCCK₁R and hCCK₁R, rat and human type 1 CCK receptors, respectively; Max, maximum; brackets denote concentration.

Fig. 2.

Agonist-induced CCK receptor internalization. A: representative confocal microscopic images demonstrating internalization of yellow fluorescent protein (YFP)-tagged CCK₁ receptors expressed on Chinese hamster ovary (CHO) cells after their occupation with nonfluorescent CCK peptides. Time points after stimulation are noted. Like CCK-8, CCK-58 stimulated prompt and extensive CCK receptor internalization, with similar patterns of distribution. The kinetics of internalization were also similar after stimulation with the 2 CCK peptides. Scale bar = 25 μm. B: quantitation of the receptor on the cell surface under these conditions. Results reflect means ± SE of data from 3 independent experiments.

Fig. 3.

CCK-58 and CCK-8 disrupt CCK₁ receptor oligomerization. Shown are bioluminescence resonance energy transfer (BRET) ratios obtained from COS cells coexpressing Renilla luciferase (Rlu)-and YFP-tagged human CCK₁ receptors (HCCKAR). Cells expressing the CCK receptor constructs were incubated with increasing concentrations of either CCK-8 or CCK-58 prior to measurement of the BRET signals. Concentration-dependent decreases in the BRET signals were observed after stimulation with each of the peptides. The shaded area represents the background BRET ratio that can be measured upon expression of structurally unrelated receptors on the cell surface or upon expression of a donor-acceptor pair with one in the plasma membrane and the other in the cytosol. Signals above this are considered to be significant. Data represent means ± SE of 4 independent experiments. *P < 0.05, **P < 0.01 compared with BRET signals obtained without exposure to CCK peptides.

Fig. 4.

Saturation BRET analysis. Shown are BRET saturation curves plotted as the ratios of YFP fluorescence to Rlu luminescence obtained when COS cells coexpressing fixed concentrations of Rlu-tagged CCK₁ receptors (donor) and increasing the concentrations of YFP-tagged CCK₁ receptors (acceptor) were exposed to the noted peptides. For control cells not exposed to CCK, the BRET ratios generated exponential curves that increased until values reached saturation, as reflected in an asymptote. In contrast, CCK-8 and CCK-58 stimulation yielded BRET saturation curves that were not statistically different from linear fits. Data are represented as means ± SE of 4 independent experiments.