Holicky, Eileen L.

Agonist-stimulated phosphorylation of guanine nucleotide-binding protein (G protein)-coupled receptors has been recognized as an important mechanism for desensitization by interfering with coupling of the activated receptor with its G protein. We recently described a mutant of the CCK receptor that modified two of five key sites of phosphorylation (S260,264A) and eliminated agonist-stimulated receptor phosphorylation, despite normal ligand binding and signaling (20). As expected, this nonphosphorylated mutant had impaired rapid desensitization but was ultimately able to be desensitized by normal receptor internalization. Here we demonstrate that this mutant receptor is also defective in resensitization, with abnormal recycling to the cell surface. To explore this, another receptor mutant was prepared, replacing the same serines with aspartates to mimic the charge of serine-phosphate (S260,264D). This mutant was expressed in a Chinese hamster ovary cell line and shown to bind CCK normally. It had accelerated kinetics of signaling and desensitization and was phosphorylated in response to agonist occupation, with all other normal sites of phosphorylation modified. It was internalized like wild-type receptors and was resensitized and trafficked normally. This provides evidence for an additional important function for phosphorylation of G protein-coupled receptors. Phosphorylation may induce a conformational change in the receptor to expose other potential sites of phosphorylation and to expose domains involved in the targeting and trafficking of endosomes. The hierarchical phosphorylation of these sites may play a key role in receptor regulation.

Differences in the molecular structure or organ distribution of receptors can limit the usefulness of a given species for drug studies. In this work, we have studied cholecystokinin (CCK) receptors in cynomolgus monkey, an animal model useful for preclinical testing. The type A CCK receptor cDNA was cloned and predicted to encode a 428 amino acid peptide that was 98% identical to the human receptor. Only 2 of the 10 residues that were distinct from the human receptor were not present in other cloned CCK receptor species. A Chinese hamster ovary cell line that stably expressed this receptor was developed. The cynomolgus receptor expressed in this environment was functionally indistinguishable from the human receptor, binding CCK with high affinity [inhibition constant ( KI) = 1.8 ± 0.5 nM] and exhibiting a potent intracellular calcium signaling response to this hormone (EC50= 6.6 ± 2.1 pM). Like the human type A CCK receptor, this receptor was expressed prominently in monkey gallbladder and stomach and was expressed in low levels in brain and pancreas. The type B CCK receptor cDNA was cloned from stomach and brain (450 residue receptor that is 96% identical to the human receptor), where it was highly expressed yet was undetectable in gallbladder or pancreas. This work confirms the relevance of the cynomolgus species for preclinical testing of drugs acting on the type A CCK receptor.

The carboxy-terminal region of many guanine nucleotide-binding protein (G protein)-coupled receptors contains important regulatory sequences such as an NP(x)2–3Y motif, a site of fatty acid acylation, and serine- and threonine-rich domains. The type A CCK receptor contains all of these, yet their significance has not been examined. We have, therefore, constructed a series of receptor site mutants and truncations that interfere with each of these motifs and expressed each in Chinese hamster ovary cells where they were studied for radioligand binding, cell signaling, receptor internalization, and intracellular trafficking. Each construct was synthesized and transported appropriately to the cell surface, where CCK bound with high affinity, elicited an inositol 1,4,5-trisphosphate response, and resulted in internalization and normal trafficking. Thus modification or elimination of each of these established sequence motifs had no substantial effect on any of these parameters of receptor and cellular function. However, an additional construct that truncated the carboxy terminus, eliminating an additional 15-amino-acid segment devoid of any currently recognized sequence motifs, resulted in a marked change in receptor trafficking, with all other parameters of receptor function normal. This mutant receptor construct was delayed at the stage of early endosomes, delaying its progress to the lysosome-enriched perinuclear compartment from the rapid time course followed by wild-type receptor and all of the other constructs. It is proposed that this region of the CCK receptor tail contains a new motif important for intracellular receptor trafficking.

Molecular mechanisms for the regulation of islet amyloid polypeptide (IAPP) gene expression remain unclear. In the present study, we investigated the effects of glucose and forskolin on IAPP gene regulation in the INS-1 islet β-cell line. Both glucose and forskolin increased the level of expression of this gene, as measured by Northern blot analysis, and increased IAPP gene transcription in a time- and concentration-dependent manner, as demonstrated in a reporter gene assay. Although inhibition of protein kinase A activity with H-89 eliminated the effect of forskolin on this gene, the glucose effect was unaffected. This supported the predominant use of a protein kinase A-independent signaling pathway for glucose regulation of the IAPP gene. Electrophoretic mobility shift assay further indicated that glucose and forskolin regulated expression of this gene by targeting different elements of the promoter. Mutation of the cAMP regulatory element flanking the IAPP coding region resulted in the loss of most of the forskolin-stimulated IAPP gene promoter activity, whereas glucose-enhanced IAPP gene transcription was unaffected. These results demonstrate parallel and distinct regulatory pathways involved in glucose- and forskolin-induced IAPP gene expression in this model β-cell system.

The targeting of lysosomal transmembrane (TM) proteins from the Golgi apparatus to lysosomes is a complex process that is only beginning to be understood. Here, the lysosomal targeting of mucolipin‐1 (Mcoln1), the TM protein defective in the autosomal recessive disease, mucolipidosis type IV, was studied by overexpressing full‐length and truncated forms of the protein in human cells, followed by detection using immunofluorescence and immunoblotting. We demonstrated that a 53‐amino acid C‐terminal region of Mcoln1 is required for efficient exit from the Golgi. Truncations lacking this region exhibited reduced delivery to lysosomes and decreased proteolytic cleavage of Mcoln1 into characteristic ∼35‐kDa fragments, suggesting that this cleavage occurs in lysosomes. In addition, we found that the co‐expression of full‐length Mcoln1 with kinase‐inactive protein kinase D (PKD) 1 or 2 inhibited Mcoln1 Golgi exit and transport to lysosomes and decreased Mcoln1 cleavage. These studies suggest that PKDs play a role in the delivery of some lysosomal resident TM proteins from the Golgi to the lysosomes.

Insulin stimulates glucose transport in fat and skeletal muscle cells primarily by inducing the translocation of GLUT4 (glucose transporter isoform 4) to the PM (plasma membrane) from specialized GSVs (GLUT4 storage vesicles). Glycosphingolipids are components of membrane microdomains and are involved in insulin-regulated glucose transport. Cellular glycosphingolipids decrease during adipocyte differentiation and have been suggested to be involved in adipocyte function. In the present study, we investigated the role of glycosphingolipids in regulating GLUT4 translocation. We decreased glycosphingolipids in 3T3-L1 adipocytes using glycosphingolipid synthesis inhibitors and investigated the effects on GLUT4 translocation using immunocytochemistry, preparation of PM sheets, isolation of GSVs and FRAP (fluorescence recovery after photobleaching) of GLUT4–GFP (green fluorescent protein) in intracellular structures. Glycosphingolipids were located in endosomal vesicles in pre-adipocytes and redistributed to the PM with decreased expression at day 2 after initiation of differentiation. In fully differentiated adipocytes, depletion of glycosphingolipids dramatically accelerated insulin-stimulated GLUT4 translocation. Although insulin-induced phosphorylation of IRS (insulin receptor substrate) and Akt remained intact in glycosphingolipid-depleted cells, both in vitro budding of GLUT4 vesicles and FRAP of GLUT4–GFP on GSVs were stimulated. Glycosphingolipid depletion also enhanced the insulin-induced translocation of VAMP2 (vesicle-associated membrane protein 2), but not the transferrin receptor or cellubrevin, indicating that the effect of glycosphingolipids was specific to VAMP2-positive GSVs. Our results strongly suggest that decreasing glycosphingolipid levels promotes the formation of GSVs and, thus, GLUT4 translocation. These studies provide a mechanistic basis for recent studies showing that inhibition of glycosphingolipid synthesis improves glycaemic control and enhances insulin sensitivity in animal models of Type 2 diabetes.

Caveolar endocytosis is an important mechanism for the uptake of certain pathogens and toxins and also plays a role in the internalization of some plasma membrane (PM) lipids and proteins. However, the regulation of caveolar endocytosis is not well understood. We previously demonstrated that caveolar endocytosis and β1-integrin signaling are stimulated by exogenous glycosphingolipids (GSLs). In this study, we show that a synthetic GSL with nonnatural stereochemistry, β-d-lactosyl-N-octanoyl-l-threo-sphingosine, (1) selectively inhibits caveolar endocytosis and SV40 virus infection, (2) blocks the clustering of lipids and proteins into GSLs and cholesterol-enriched microdomains (rafts) at the PM, and (3) inhibits β1-integrin activation and downstream signaling. Finally, we show that small interfering RNA knockdown of β1 integrin in human skin fibroblasts blocks caveolar endocytosis and the stimulation of signaling by a GSL with natural stereochemistry. These experiments identify a new compound that can interfere with biological processes by inhibiting microdomain formation and also identify β1 integrin as a potential mediator of signaling by GSLs.

Sphingolipids (SLs) play important roles in membrane structure and cell function. Here, we examine the SL requirements of various endocytic mechanisms using a mutant cell line and pharmacological inhibitors to disrupt SL biosynthesis. First, we demonstrated that in Chinese hamster ovary cells we could distinguish three distinct mechanisms of clathrin-independent endocytosis (caveolar, RhoA, and Cdc42 dependent) which differed in cargo, sensitivity to pharmacological agents, and dominant negative proteins. General depletion of SLs inhibited endocytosis by each clathrin-independent mechanism, whereas clathrin-dependent uptake was unaffected. Depletion of glycosphingolipids (GSLs; a subgroup of SLs) selectively blocked caveolar endocytosis and decreased caveolin-1 and caveolae at the plasma membrane. Caveolar endocytosis and PM caveolae could be restored in GSL-depleted cells by acute addition of exogenous GSLs. Disruption of RhoA- and Cdc42-regulated endocytosis by SL depletion was shown to be related to decreased targeting of these Rho proteins to the plasma membrane and could be partially restored by exogenous sphingomyelin but not GSLs. Both the in vivo membrane targeting and in vitro binding to artificial lipid vesicles of RhoA and Cdc42 were shown to be dependent upon sphingomyelin. These results provide the first evidence that SLs are differentially required for distinct mechanisms of clathrin-independent endocytosis.

Abstract Glycosphingolipids are known to play roles in integrin-mediated cell adhesion and migration; however, the mechanisms by which glycosphingolipids affect integrins are unknown. Here, we show that addition of the glycosphingolipid, C8-lactosylceramide (C8-LacCer), or free cholesterol to human fibroblasts at 10°C causes the formation of glycosphingolipid-enriched plasma membrane domains as shown by visualizing a fluorescent glycosphingolipid probe, BODIPY-LacCer, incorporated into the plasma membrane of living cells. Addition of C8-LacCer or cholesterol to cells initiated the clustering of β1-integrins within these glycosphingolipid-enriched domains and the activation of the β1-integrins as assessed using a HUTS antibody that only binds activated integrin. On warming to 37°C, β1-integrins were rapidly internalized via caveolar endocytosis in cells treated with C8-LacCer or cholesterol, whereas little β1-integrin was endocytosed in untreated fibroblasts. Incubation of cells with C8-LacCer or cholesterol followed by warm-up caused src activation, a reorganization of the actin cytoskeleton, translocation of RhoA GTPase away from the plasma membrane as visualized using total internal reflection fluorescence microscopy, and transient cell detachment. These studies show that LacCer can regulate integrin function both by modulating integrin clustering in microdomains and by regulating integrin endocytosis via caveolae. Our findings suggest the possibility that aberrant levels of glycosphingolipids found in cancer cells may influence cell attachment events by direct effects on integrin clustering and internalization.