Abstract
The lipid microenvironment of receptors can influence their conformation, function, and regulation. Cholecystokinin (CCK)-stimulated signaling is abnormal in some forms of hyperlipidemia, suggesting the possibility of unique sensitivity to its lipid environment. Here we examined the influence of cholesterol and sphingolipids on CCK receptors in model Chinese hamster ovary cell systems having lipid levels modified. Cholesterol was modulated chemically or metabolically, and sphingolipids were modulated using a temperature-sensitive cell line (SPB-1). Receptor conformation was probed with a fluorescent full agonist ligand, Alexa 488-conjugated Gly-[Nle(28,31)]CCK-(26-33), shown previously to decrease in anisotropy and lifetime when occupying a receptor in the active conformation (Harikumar, K. G., Pinon, D. L., Wessels, W. S., Prendergast, F. G., and Miller, L. J. (2002) J. Biol. Chem. 277, 18552-18560). Anisotropy and lifetime of this probe were increased and prolonged with cholesterol enrichment, and decreased and shortened with depletion of cholesterol or sphingolipids. The increase in these parameters with cholesterol enrichment may reflect change in CCK receptor conformation toward its inactive, uncoupled state. Indeed, cholesterol enrichment resulted in nonproductive agonist ligand binding, with affinity of binding higher than normal and calcium signaling in response to this reduced. In cholesterol- and sphingolipid-depleted states, the receptor moved into conformations that were less than optimal. With cholesterol depletion, both ligand binding and signaling were decreased, yet internalization and trafficking were unperturbed. With sphingolipid depletion, ligand binding and signaling were normal, but internalization and trafficking were markedly inhibited. Of note, normal transferrin receptor trafficking through the same clathrin-dependent pathway was maintained under these conditions. Thus, lipid microenvironment of the CCK receptor is particularly important, with different lipids having distinct effects.
Highlights
¶ Present address: Dept. of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo 1628640, Japan
The results showed that ϳ90% of internalized CCK receptors were able to recycle to the cell surface of SPB-1-CCKR cells grown at the nonpermissive temperature in the presence of sphingomyelin, whereas this was quite impaired when these cells were deprived of sphingolipids
G protein-coupled receptors were believed to function as single molecules, only interacting with their heterotrimeric G protein proximal effectors after agonist occupation
Summary
Despite these effects on agonist binding and signaling, the internalization and trafficking of the CCK receptor were unperturbed with cholesterol depletion. This cell line has been fully characterized previously, establishing the expression of fully functional receptors that bind CCK, signal, and are internalized in a normal manner [16]. Characterization of CCK Receptor Function in Different Cellular Lipid Environments—Cholesterol depletion either with MCD or by growing the CHO-CCKR cells in LPDS medium supplemented with metabolic inhibitors resulted in a shift to lower affinity binding of CCK.
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