Eileen L. Holicky

Relationship between native and recombinant cholecystokinin receptors: role of differential glycosylation.

&NA; In an attempt to establish the relationship between the protein encoded by the recently cloned type A cholecystokinin (CCK) receptor cDNA and the two distinct plasmalemmal proteins on the rat pancreatic acinar cell that were previously described as candidates to represent this receptor, we have established a Chinese hamster ovary (CHO) cell line stably expressing large amounts of this recombinant protein and have used biochemical methods to characterize it directly. Upon affinity labeling, this protein migrated faster on a sodium do-decyl sulfate-polyacrylamide gel than the Mr 85,000–95,000 molecule previously felt to represent the best candidate. However, deglycosylation with endoglycosi-dase F demonstrated that it had the same size core protein as that candidate, and this identification was further supported by protease peptide mapping. We postulated that the structural differences between the recombinant and the native proteins related to differences in glycosylation. Consistent with this, lectin-binding experiments demonstrated that both represented complex glycoproteins but that only the native receptor-bound Ulex europeus agglutinin I. Since this lectin binds to fucose residues that are added late in glycoprotein biosynthesis, it is possible that the distinct processing observed affected only that step. In spite of this structural difference, the type A CCK receptor-bearing CHO cell CCK receptor was functionally indistinguishable from the native acinar cell receptor. This included its ability to initiate signaling cascades, its sensitivity to stable GTP analogues, and its binding affinities for agonists and antagonists. The fidelity of this receptor expression system, while representing a 25-fold increase in receptor density over the native pancreatic acinar cell, should provide an ideal substrate for the examination of structure-function relationships within this molecule.

Abnormal processing of the human cholecystokinin receptor gene in association with gallstones and obesity

BACKGROUND & AIMS Cholesterol gallstone disease and obesity are often associated and share the potential, yet unreported, common etiology of cholecystokinin (CCK) dysfunction. While cloning the human CCK-A receptor complementary DNA (cDNA), we found predominance of a 262-base pair coding region deletion in a cDNA library prepared from a patient with this phenotype. The aim of this study was to determine the abundance, functional significance, and mechanism for generating this gene product. METHODS Relative abundance of CCK receptor gene products was determined using polymerase chain reaction and hybridization analysis. Constructs were expressed in COS cells and studied for radioligand binding and intracellular calcium responses. A human genomic clone for this receptor was sequenced, and the critical regions were compared with those of the patient. RESULTS Ninety-three percent of the patients CCK receptor transcripts contained the 262-base pair deletion, whereas only 1.5% +/- 0.9% of control patients had the deletion. This encoded a receptor that did not bind or signal. The deletion corresponded with the third exon; however, this sequence and flanking introns were normal in the patient. CONCLUSIONS Abnormality of processing an apparently normal CCK receptor gene yields the predominant product with an absent third exon and encoding a nonfunctional receptor, probably reflecting a defective trans-acting splicing factor. An atypical lariat region in the third intron may explain the presence of small amounts of this product in control patients.

Protein transduction of Rab9 in Niemann-Pick C cells reduces cholesterol storage

Niemann‐Pick disease type C (NPC) is a genetic disorder in which patient cells exhibit lysosomal accumulation of cholesterol and sphingolipids (SLs) caused by defects in either NPC1 or NPC2 proteins. We previously demonstrated that NPC1 human skin fibroblasts overexpressing endosomal Rab proteins (Rab7 or Rab9) showed a correction in the storage disease phenotype. In the current study, we used protein transduction to further investigate Rab9‐mediated reduction of stored lipids in NPC cells. Recombinant human Rab9 fused with the herpes simplex virus VP22 protein fragment was overexpressed, purified, and added to culture medium to induce protein transduction. When VP22‐Rab9 was transduced into NPC1 fibroblasts, nearly all cells showed significant reduction in cellular free cholesterol levels, with no cytotoxicity up to 5 µM. A fraction of the VP22‐Rab9 that was transduced into the cells was shown to bind to rab GDP dissociation inhibitor, suggesting that this pool of VP22‐Rab9 had become prenylated. The reduction in cellular free cholesterol was associated with correction of abnormal intracellular trafficking of BODIPY‐lactosylceramide and an increase of sterols in the culture media. The clearance of lysosomal free cholesterol was also associated with a decrease in LDL‐receptor levels. In addition, we demonstrated reduction of intracellular cholesterol by VP22‐Rab9 transduction in NPC2 fibroblasts and in cultured mouse NPC1 neurons. These observations provide important new information about the correction of membrane traffic in NPC cells by Rab9 overexpression and may lead to new therapeutic approaches for treatment of this disease.

The Glycosphingolipid, Lactosylceramide, Regulates β1-Integrin Clustering and Endocytosis

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 degrees 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 beta1-integrins within these glycosphingolipid-enriched domains and the activation of the beta1-integrins as assessed using a HUTS antibody that only binds activated integrin. On warming to 37 degrees C, beta1-integrins were rapidly internalized via caveolar endocytosis in cells treated with C8-LacCer or cholesterol, whereas little beta1-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.

Heterodimerization of Type A and B Cholecystokinin Receptors Enhance Signaling and Promote Cell Growth

Dimerization of several G protein-coupled receptors has recently been described, but little is known about its clinical and functional relevance. Cholecystokinin (CCK) and gastrin are structurally related gastrointestinal and neuronal peptides whose functions are mediated by two structurally related receptors in this superfamily, the type A and B CCK receptors. We previously demonstrated spontaneous homodimerization of type A CCK receptors and the dissociation of those complexes by agonist occupation (Cheng, Z. J., and Miller, L. J. (2001) J. Biol. Chem. 276, 48040-48047). Here, for the first time, we also demonstrate spontaneous homodimerization of type B CCK receptors, as well as heterodimerization of that receptor with the type A CCK receptor. Unlike type A CCK receptor dimers, the homodimerization of type B CCK receptors was not affected by ligand occupation. However, although heterodimers of type A and B CCK receptors bound natural agonists normally, they exhibited unusual functional and regulatory characteristics. Such complexes demonstrated enhanced agonist-stimulated cellular signaling and delayed agonist-induced receptor internalization. As a likely consequence, agonist-stimulated cell growth was markedly enhanced in cells simultaneously expressing both of these receptors. Our results provide the first evidence that heterodimerization of G protein-coupled receptors can form a more “powerful” signaling unit, which has potential clinical significance in promoting cell growth.

Identification of an Interaction between Residue 6 of the Natural Peptide Ligand and a Distinct Residue within the Amino-terminal Tail of the Secretin Receptor

Photoaffinity labeling is a powerful tool for the characterization of the molecular basis of ligand binding. We recently used this technique to demonstrate the proximity between a residue within the carboxyl-terminal half of a secretin-like ligand and the amino-terminal domain of the secretin receptor (Dong, M., Wang, Y., Pinon, D. I., Hadac, E. M., and Miller, L. J. (1999)J. Biol. Chem. 274, 903–909). In this work, we have developed another novel radioiodinatable secretin analogue ([Bpa6,Tyr10]rat secretin-27) that incorporates a photolabilep-benzoyl-l-phenylalanine (Bpa) residue into position 6 of the amino-terminal half of the ligand and used this to identify a specific receptor residue proximate to it. This probe specifically bound to the secretin receptor with high affinity (IC50 = 13.2 ± 2.5 nm) and was a potent stimulant of cAMP accumulation in secretin receptor-bearing Chinese hamster ovary-SecR cells (EC50 = 720 ± 230 pm). It covalently labeled the secretin receptor in a saturable and specific manner. Cyanogen bromide cleavage of this molecule yielded a single labeled fragment that migrated on an SDS-polyacrylamide gel at M r = 19,000 that shifted to 10 after deglycosylation, most consistent with either of two glycosylated fragments within the amino-terminal tail. By immunoprecipitation with antibody directed to epitope tags incorporated into each of the two candidate fragments, the most distal fragment at the amino terminus was identified as the domain of labeling. The labeled domain was further refined to the first 16 residues by endoproteinase Lys-C cleavage and by cyanogen bromide cleavage of another receptor construct in which Val16 was mutated to Met. Radiochemical sequencing of photoaffinity-labeled secretin receptor fragments established that Val4 was the specific site of covalent attachment. This provides the first residue-residue contact between a secretin ligand and its receptor and will contribute substantially to the molecular understanding of this interaction.

Inhibition of caveolar uptake, SV40 infection, and β1-integrin signaling by a nonnatural glycosphingolipid stereoisomer

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.

Caveolar endocytosis and microdomain association of a glycosphingolipid analog is dependent on its sphingosine stereochemistry

We have previously shown that glycosphingolipid analogs are internalized primarily via caveolae in various cell types. This selective internalization was not dependent on particular carbohydrate headgroups or sphingosine chain length. Here, we examine the role of sphingosine structure in the endocytosis of BODIPY™-tagged lactosylceramide (LacCer) analogs via caveolae. We found that whereas the LacCer analog with the natural (d-erythro) sphingosine stereochemistry is internalized mainly via caveolae, the non-natural (l-threo) LacCer analog is taken up via clathrin-, RhoA-, and Cdc42-dependent mechanisms and largely excluded from uptake via caveolae. Unlike the d-erythro-LacCer analog, the l-threo analog did not cluster in membrane microdomains when added at higher concentrations (5–20 μm). In vitro studies using small unilamellar vesicles and giant unilamellar vesicles demonstrated that l-threo-LacCer did not undergo a concentration-dependent excimer shift in fluorescence emission such as that seen with BODIPY™-sphingolipids with natural stereochemistry. Molecular modeling studies suggest that in d-erythro-LacCer, the disaccharide moiety extends above and in the same plane as the sphingosine hydrocarbon chain, while in l-threo-LacCer the carbohydrate group is nearly perpendicular to the hydrocarbon chain. Together, these results suggest that the altered stereochemistry of the sphingosine group in l-threo-LacCer results in a perturbed structure, which is unable to pack closely with natural membrane lipids, leading to a reduced inclusion in plasma membrane microdomains and decreased uptake by caveolar endocytosis. These findings demonstrate the importance of the sphingolipid stereochemistry in the formation of membrane microdomains.

Development of a Rab9 Transgenic Mouse and Its Ability to Increase the Lifespan of a Murine Model of Niemann-Pick Type C Disease

Niemann-Pick, type C (NP-C) disease is an autosomal recessive neurovisceral storage disorder in which cholesterol and sphingolipids accumulate. There is no specific treatment for this disease, which is characterized by progressive neurological deterioration, sometimes accompanied by hepatosplenomegaly. We and others have shown that overexpression of certain Rab GTPases corrects defective membrane trafficking and reduces lipid storage in cultured NP-C fibroblasts. Here, we tested the possibility that Rab protein overexpression might also have beneficial effects in vivo using a murine model of NP-C. We first generated several lines of transgenic mice that ubiquitously overexpress Rab9 up to approximately 30-fold more than endogenous levels and found that the transgene expression had no obvious effects on fertility, behavior, or lifespan in normal mice. These transgenic strains were then crossed with NP-C mutant mice to produce NP-C homozygous recessive mice with and without the Rab9 transgene. Life expectancy of the NPC1 homozygous recessive animals was extended up to 22% depending on gender and the transgenic strain that was used. Histological studies and lipid analysis of brain sections indicated that the NP-C mice carrying the Rab9 transgene had dramatically reduced storage of GM(2) and GM(3) gangliosides relative to NP-C animals lacking the transgene. These results demonstrate that Rab9 overexpression has the potential to reduce stored lipids and prolong lifespan in vivo.

Androgen receptor binding activity in human prostate cancer.

Androgen binding (cytosol and nucleus) was measured in tissue obtained from 223 untreated patients with proven prostate cancer (199 primary tumor, 24 malignant lymph nodes), 19 patients with hormone refractory cancer, and 46 patients with benign prostatic hyperplasia (BPH). The mean binding in both the cytosol and nucleus was significantly higher for patients with cancer than for those with BPH. Binding appeared to correlate with tumor stage. Androgen binding in malignant nodes can differ from that in the primary tissue and can vary from node to node in the same patient. Results obtained from an assay using a single saturating concentration of R1881 correlated well with those calculated from a full six‐point Scatchard analysis when an adequate amount (500 mg) of tissue was available. However, binding results obtained from a single‐point analysis performed on needle biopsy specimens (about 50 mg) obtained before complete surgical removal of the prostate correlated poorly with those derived from a full six‐point analysis performed on tissue (500–1000 mg) removed from the center of the malignancy. Androgen binding in nuclear extracts of histologically benign tissue adjacent to the malignancy was significantly higher than in nuclear extracts of BPH tissue. Cytosolic androgen binding in tissue removed from patients who were refractory to hormonal therapy was higher than in tissue from untreated cancer patients. The binding of estradiol by extracts of benign and malignant prostate tissue was low or absent and, thus, did not appear to be a significant phenomenon.