Cardiac myocyte adenosine receptors and caveolae.

The purine nucleoside adenosine exerts numerous effects in the mammalian heart, the most well-recognized being regulation of coronary blood flow and cardiac conduction. These effects are mediated via activation of G protein linked adenosine receptor subtypes, A(2a) and A(1) receptors, located primarily on vascular cells and cardiac myocytes, respectively. Although adenosine A(1) receptors are also expressed in ventricular myocytes, adenosine exerts no significant direct effects in these cells. A recent report from our laboratory indicates that ventricular myocyte A(1) receptors are concentrated in caveolin enriched plasma membrane microdomains referred to as caveolae. This review focuses on these recent findings and their relevance to subcellular compartmentalization of A(1) receptor signaling in ventricular myocardium.

Caveolin-1 negatively regulates SR-BI mediated selective uptake of high-density lipoprotein-derived cholesteryl ester.

The class B, type I scavenger receptor (SR-BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl esters and the efflux of free cholesterol. SR-BI is predominantly associated with caveolae in Chinese hamster ovary cells. The caveola protein, caveolin-1, binds to cholesterol and is involved in intracellular cholesterol trafficking. We previously demonstrated a correlative increase in caveolin-1 expression and the selective uptake of HDL cholesteryl esters in phorbol ester-induced differentiated THP-1 cells. The goal of the present study was to determine if the expression of caveolin-1 is the causative factor in increasing selective cholesteryl ester uptake in macrophages. To test this, we established RAW and J-774 cell lines that stably expressed caveolin-1. Transfection with caveolin-1 cDNA did not alter the amount of 125I-labeled HDL that associated with the cells, although selective uptake of HDL [3H]cholesteryl ether was decreased by approximately 50%. The amount of [3H]cholesterol effluxed to HDL was not affected by caveolin-1. To directly address whether caveolin-1 inhibits SR-BI-dependent selective cholesteryl ester uptake, we overexpressed caveolin-1 by adenoviral vector gene transfer in Chinese hamster ovary cells stably transfected with SR-BI. Caveolin-1 inhibited the selective uptake of HDL [3H]cholesteryl ether by 50-60% of control values without altering the extent of cell associated HDL. We next used blocking antibodies to CD36 and SR-BI to demonstrate that the increase in selective [3H]cholesteryl ether uptake previously seen in differentiated THP-1 cells was independent of SR-BI. Finally, we used beta-cyclodextrin and caveolin overexpression to demonstrate that caveolae depleted of cholesterol facilitate SR-BI-dependent selective cholesteryl ester uptake and caveolae containing excess cholesterol inhibit uptake. We conclude that caveolin-1 is a novel negative regulator of SR-BI-dependent selective cholesteryl ester uptake.

Influence of caveolin, cholesterol, and lipoproteins on nitric oxide synthase: implications for vascular disease.

Caveolin-1 traffics cholesterol between the endoplasmic reticulum and cell surface caveolae in a non-vesicle chaperone complex which contains heat shock protein 56, cyclophilin 40, and cyclophilin A. Recent studies demonstrate that endothelial nitric oxide synthase (eNOS), caveolin, hetero-trimeric G-protein coupled receptors, and a calcium channel form an activation complex that is associated with cholesterol-rich caveolae. Oxidized LDL depletes caveolae of cholesterol and prevents agonist stimulation of eNOS by disrupting the activation complex. HDL antagonizes the effects of oxLDL by donating cholesterol to caveolae, thereby preserving the structure and function of caveolae. These findings and others provide a possible mechanistic basis for some of the molecular changes observed in vascular disease.

The role of caveolae and caveolin in vesicle-dependent and vesicle-independent trafficking.

Caveolae can mediate endocytosis, transcytosis, and potocytosis. Our understanding of these processes as well as the elucidation of the molecular machinery involved has greatly expanded. In addition, caveolin, a 22 kDa protein often associated with caveolae, can promote the trafficking of sterol through the cytoplasm independent of vesicles. Caveolin also influences the formation, morphology, and function of caveolae. The ability of caveolae and caveolin to mediate macromolecular transport directly impacts a variety of physiological and pathophysiological processes.

17beta-Estradiol promotes the up-regulation of SR-BII in HepG2 cells and in rat livers.

The scavenger receptor class B type I (SR-BI) binds to HDL and mediates the selective uptake of cholesterol esters from HDL to cells. SR-BII is an alternatively spliced product of the SR-BI gene that only differs in the C-terminal cytoplasmic domain. Previous studies with male mice demonstrated that SR-BII comprises about 12% of the total SR-BI/SR-BII present in liver. In the current studies we used a liver cell line, HepG2, and a rat estrogen replacement model to examine the effects of estrogen on the expression of SR-BII. HepG2 cells express SR-BI but not SR-BII. SR-BI/SR-BII - blocking antibodies demonstrated that HepG2 cells selectively internalize cholesterol esters in a SR-BI - dependent manner. Incubation of HepG2 cells with 10 pM of 17beta-estradiol for 12 h eliminated the expression of SR-BI and promoted the up-regulation of SR-BII. Radiolabeled HDL-binding studies demonstrated that 17beta-estradiol increased the number of HDL binding sites by 3-fold in HepG2 cells. However, 17beta-estradiol - treated cell internalized approximately 25% less cholesterol ester than vehicle-only-treated cells. The livers obtained from male rats and ovariectomized female rats contained SR-BI and a small amount of SR-BII. In contrast, the livers obtained from intact female rats and ovariectomized female rats receiving estrogen replacement contained SR-BII and a small amount of SR-BI. The amount of SR-BI and SR-BII in adrenal tissue was not affected by any of the experimental treatments. We conclude that estrogen up-regulates SR-BII in HepG2 cells and rat liver.

Palmitoylation of caveolin-1 is required for cholesterol binding, chaperone complex formation, and rapid transport of cholesterol to caveolae.

We previously demonstrated that a caveolin-chaperone complex transports newly synthesized cholesterol from the endoplasmic reticulum through the cytoplasm to caveolae. Caveolin-1 has a 33-amino acid hydrophobic domain and three sites of palmitoylation in proximity to the hydrophobic domain. In the present study, we hypothesized that palmitoylation of caveolin-1 is necessary for binding of cholesterol, formation of a caveolin-chaperone transport complex, and rapid, direct transport of cholesterol to caveolae. To test this hypothesis, four caveolin-1 constructs were generated that substituted an alanine for a cysteine at position 133, 143, or 156 or all three sites (triple mutant). These mutated caveolins and wild type caveolin-1 were stably expressed in the lymphoid cell line, L1210-JF, which does not express caveolin-1, does not form a caveolin-chaperone complex, and does not transport newly synthesized cholesterol to caveolae. All of the caveolins were expressed and the proteins localized to plasma membrane caveolae. Wild type caveolin-1 and mutant 133 assembled into complete transport complexes and rapidly (10-20 min) transported cholesterol to caveolae. Caveolin mutants 143 and 156 did not assemble into complete transport complexes, weakly associated with cholesterol, and transported small amounts of cholesterol to caveolae. The triple mutant did not assemble into complete transport complexes and did not associate with cholesterol. We conclude that palmitoylation of caveolin-1 at positions 143 and 156 is required for cholesterol binding and transport complex formation.

Activated cardiac adenosine A(1) receptors translocate out of caveolae.

The cardiac affects of the purine nucleoside, adenosine, are well known. Adenosine increases coronary blood flow, exerts direct negative chronotropic and dromotropic effects, and exerts indirect anti-adrenergic effects. These effects of adenosine are mediated via the activation of specific G protein-coupled receptors. There is increasing evidence that caveolae play a role in the compartmentalization of receptors and second messengers in the vicinity of the plasma membrane. Several reports demonstrate that G protein-coupled receptors redistribute to caveolae in response to receptor occupation. In this study, we tested the hypothesis that adenosine A(1) receptors would translocate to caveolae in the presence of agonists. Surprisingly, in unstimulated rat cardiac ventricular myocytes, 67 +/- 5% of adenosine A(1) receptors were isolated with caveolae. However, incubation with the adenosine A(1) receptor agonist 2-chlorocyclopentyladenosine induced the rapid translocation of the A(1) receptors from caveolae into non-caveolae plasma membrane, an effect that was blocked by the adenosine A(1) receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine. An adenosine A(2a) receptor agonist did not alter the localization of A(1) receptors to caveolae. These data suggest that the translocation of A(1) receptors out of caveolae and away from compartmentalized signaling molecules may explain why activation of ventricular myocyte A(1) receptors are associated with few direct effects.

Effects of diary food supplements on bone mineral density in teenage girls.

BACKGROUND: Bone mineral density (BMD) is largely genetically determined and this influence is most powerful in the period of rapid skeletal development in childhood and late adolescence but environmental factors such as exercise and dietary calcium intake may influence up to 20%. AIMS OF THE STUDY: The aims of the study were to examine healthy late adolescent females for the effects and benefits of a high calcium intake from dairy product foods on bone mineral density, body composition, lipids and biochemistry. The secondary aim is determine whether a high intake of dairy product foods in the diet is acceptable for this age group long term. METHODS: Ninety-one teenage girls who participated in a two-year randomised controlled study on the effect of dairy food supplementation on dietary patterns, body composition and bone density in post-pubertal teenage girls were approached one year after the cessation of the study to determine the effects of the cessation of dairy supplements on bone mineral density, dietary habits, biochemical markers, body composition and blood lipids. Bone mineral density and bone mineral content were assessed at the hip, spine and total body. Anthropometric data were collected, and exercise, Tanner, dietary assessment, preference and compliance questionnaires were administered. Lipid profiles, hydroxyproline excretion and urinary calcium and sodium excretion measurements were performed. RESULTS: There were no significant differences between the 2 groups for height, weight, lean and fat mass. The supplemented group had significantly higher calcium, phosphorus and protein intake during the supplementation period (p < 0.001). No differences were seen between the groups 12 months after supplementation finished. There were no significant differences in exercise level, preference or acceptability of dairy products or in the lipids and bone markers between baseline the end of supplementation and 1 year follow-up. There was a significant increase in trochanter (4.6%), lumbar spine (1.5%) and femoral neck (4.8%) BMD (p < 0.05) in the high calcium group at the end of supplementation. There was an increase in bone mineral content at the trochanter (p < 0.05) and lumbar spine; however the latter was not statistically significant, in the high calcium group at the end of supplementation. There was no difference in vertebral height or width at any stage of the study, indicating no influence on bone size. CONCLUSIONS: In this 3 year study (2 years of supplementation, 1 year follow-up), teenage girls, aged 15-18 years, were able to significantly increase their BMD at the trochanter, femoral neck and lumbar spine when supplemented with dairy product foods to a mean calcium intake of 1160 mg/d. There was also an effect seen on the BMC particularly at the trochanter and to a lesser extent at the lumbar spine. The dietary calcium intake achieved did not adversely affect body weight, fat and lean mass or blood lipid profiles. Twelve months after the supplementation finished the girls had returned to their baseline diet, indicating self-selection of a high dairy product diet may be hard to achieve.

High density lipoprotein prevents oxidized low density lipoprotein-induced inhibition of endothelial nitric-oxide synthase localization and activation in caveolae.

Oxidized LDL (oxLDL) depletes caveolae of cholesterol, resulting in the displacement of endothelial nitric-oxide synthase (eNOS) from caveolae and impaired eNOS activation. In the present study, we determined if the class B scavenger receptors, CD36 and SR-BI, are involved in regulating nitric-oxide synthase localization and function. We demonstrate that CD36 and SR-BI are expressed in endothelial cells, co-fractionate with caveolae, and co-immunoprecipitate with caveolin-1. Co-incubation of cells with 10 microgram/ml high density lipoprotein (HDL) prevented oxLDL-induced translocation of eNOS from caveolae and restored acetylcholine-induced nitric-oxide synthase stimulation. Acetylcholine caused eNOS activation in cells incubated with 10 microgram/ml oxLDL (10-15 thiobarbituric acid-reactive substances) and blocking antibodies to CD36, whereas cells treated with only oxLDL were unresponsive. Furthermore, CD36-blocking antibodies prevented oxLDL-induced redistribution of eNOS. SR-BI-blocking antibodies were used to demonstrate that the effects of HDL are mediate by SR-BI. HDL binding to SR-BI maintained the concentration of caveola-associated cholesterol by promoting the uptake of cholesterol esters, thereby preventing oxLDL-induced depletion of caveola cholesterol. We conclude that CD36 mediates the effects of oxLDL on caveola composition and eNOS activation. Furthermore, HDL prevents oxLDL from decreasing the capacity for eNOS activation by preserving the cholesterol concentration in caveolae and, thereby maintaining the subcellular location of eNOS.

Oxidized low density lipoprotein displaces endothelial nitric-oxide synthase (eNOS) from plasmalemmal caveolae and impairs eNOS activation.

Hypercholesterolemia-induced vascular disease and atherosclerosis are characterized by a decrease in the bioavailability of endothelium-derived nitric oxide. Endothelial nitric-oxide synthase (eNOS) associates with caveolae and is directly regulated by the caveola protein, caveolin. In the present study, we examined the effects of oxidized low density lipoprotein (oxLDL) on the subcellular location of eNOS, on eNOS activation, and on caveola cholesterol in endothelial cells. We found that treatment with 10 microgram/ml oxLDL for 60 min caused greater than 90% of eNOS and caveolin to leave caveolae. Treatment with oxLDL also inhibited acetylcholine-induced activation of eNOS but not prostacyclin production. oxLDL did not affect total cellular eNOS abundance. Oxidized LDL also did not affect the palmitoylation, myristoylation or phosphorylation of eNOS. Oxidized LDL, but not native LDL, or HDL depleted caveolae of cholesterol by serving as an acceptor for cholesterol. Cyclodextrin also depleted caveolae of cholesterol and caused eNOS and caveolin to translocate from caveolae. Furthermore, removal of oxLDL allowed eNOS and caveolin to return to caveolae. We conclude that oxLDL-induced depletion of caveola cholesterol causes eNOS to leave caveolae and inhibits acetylcholine-induced activation of the enzyme. This process may be an important mechanism in the early pathogenesis of atherosclerosis.

Macrophage scavenger receptors and foam cell formation.

Scavenger receptors bind and internalize modified low-density lipoprotein (LDL) and high-density lipoprotein (HDL). Because the expression of scavenger receptors is not down-regulated by cholesterol, macrophages (Mphi) expressing scavenger receptors can internalize substantial quantities of cholesteryl ester from oxidized LDL and HDL, leading to foam cell formation. Mphi express several different classes of the growing scavenger receptor family on their cell surface and their relative contribution to Mphi cholesterol physiology and atherogenesis is the subject of intense investigation. We focus on the potential role of two scavenger receptors, macrosialin and SR-BI/II in Mphi cholesterol metabolism. Macrosialin is a predominantly Mphi-specific oxidized LDL-binding protein and an atherogenic diet markedly up-regulates its hepatic expression in atherosclerosis-susceptible and atherosclerosis-resistant mouse strains. The HDL receptor, SR-BI and its splicing variant SR-BII, colocalize with caveolin in caveolae in Mphi. Caveolae are initial acceptor sites for cholesteryl esters and these findings indicate a possible role for caveolae and SR-BI in Mphi-selective lipid uptake and in regulating Mphi cholesterol flux in the vascular wall.

Co-expression of scavenger receptor-BI and caveolin-1 is associated with enhanced selective cholesteryl ester uptake in THP-1 macrophages.

Scavenger receptor (SR)-BI mediates the selective uptake of high density lipoprotein (HDL) cholesteryl esters and the efflux of free cholesterol. In Chinese hamster ovary (CHO) cells, SR-BI is predominantly associated with caveolae which we have recently demonstrated are the initial loci for membrane transfer of HDL cholesteryl esters. Because cholesterol accumulation in macrophages is a critical event in atherogenesis, we investigated the expression of SR-BI and caveolin-1 in several macrophage cell lines. Human THP-1 monocytes were examined before and after differentiation to macrophages by treatment with 200 nm phorbol ester for 72 h. Undifferentiated THP-1 cells expressed caveolin-1 weakly whereas differentiation up-regulated caveolin-1 expression greater than 50-fold. In contrast, both undifferentiated and differentiated THP-1 cells expressed similar levels of SR-BI. Differentiation of THP-1 cells increased the percent of membrane cholesterol associated with caveolae from 12% +/- 1.9% to 38% +/- 3.1%. The increase in caveolin-1 expression was associated with a 2- to 3-fold increase in selective cholesterol ether uptake from HDL. Two mouse macrophage cell lines, J774 and RAW, expressed levels of SR-BI similar to differentiated THP-1 cells but did not express detectable levels of caveolin-1. In comparison to differentiated THP-1 cells, RAW and J774 cells internalized 9- to 10-fold less cholesteryl ester. We conclude that differentiated THP-1 cells express both caveolin-1 and SR-BI and that their co-expression is associated with enhanced selective cholesteryl ester uptake.

The prevalence of malnutrition in elderly hip fracture patients.

AIM: To determine the prevalence of protein and energy malnutrition in elderly patients with a fracture of the proximal femur, in New Zealand. METHODS: Consecutive elderly patients (65 years and over) admitted to Christchurch Hospital with a fracture of the proximal femur over a four-month period were recruited. Nutritional indices were measured within three days of admission. These included triceps skinfold thicknesses, mid upper arm circumference, serum albumin and pre-albumin. RESULTS: Forty-two per cent of patients had at least two, and nine per cent had three, indicators of protein and energy malnutrition present on admission. There was no significant difference in the prevalence of malnutrition between young old (<80 years) and old old (80 years and over) patients. Patients residing in an institution had lower mean protein reserves, as indicated by lower corrected arm muscle area (p=0.003) and pre-albumin levels (p=0.09), than those living in the community. A drink, rather than a pudding or biscuit, was the preferred protein and energy supplement form. Ensure Plus (lactose-free) and Fortisip (lactose-free) were the most preferred drink supplements. CONCLUSION: Protein and energy malnutrition is common in elderly New Zealanders who fracture their femur. The prevalence is comparable to overseas data. These patients prefer nutritional supplementation given as a drink.

The class B, type I scavenger receptor promotes the selective uptake of high density lipoprotein cholesterol ethers into caveolae.

The uptake of cholesterol esters from high density lipoproteins (HDLs) is characterized by the initial movement of cholesterol esters into a reversible plasma membrane pool. Cholesterol esters are subsequently internalized to a nonreversible pool. Unlike the uptake of cholesterol from low density lipoproteins, cholesterol ester uptake from HDL does not involve the internalization and degradation of the particle and is therefore termed selective. The class B, type I scavenger receptor (SR-BI) has been identified as an HDL receptor and shown to mediate selective cholesterol ester uptake. SR-BI is localized to cholesterol- and sphingomyelin-rich microdomains called caveolae. Caveolae are directly involved in cholesterol trafficking. Therefore, we tested the hypothesis that caveolae are acceptors for HDL-derived cholesterol ether (CE). Our studies demonstrate that in Chinese hamster ovary cells expressing SR-BI, >80% of the plasma membrane associated CE is present in caveolae after 7.5 min of selective cholesterol ether uptake. We also show that excess, unlabeled HDL can extract the radiolabeled CE from caveolae, demonstrating that caveolae constitute a reversible plasma membrane pool of CE. Furthermore, 50% of the caveolae-associated CE can be chased into a nonreversible pool. We conclude that caveolae are acceptors for HDL-derived cholesterol ethers, and that caveolae constitute a reversible, plasma membrane pool of cholesterol ethers.

Class B scavenger receptors, caveolae and cholesterol homeostasis.

Class B scavenger receptors are predominantly localized to cholesterol and sphingomyelin-enriched domains within the plasma membrane, called caveolae. Caveolae and their associated protein, caveolin, have been implicated in cholesterol trafficking and in the regulation of cellular cholesterol homeostasis. Recent studies indicate that scavenger receptor, class B, type I (SR-BI) mediates cholesterol flux between cells and lipoproteins. Caveolae appear to be the sites within the plasma membrane where such exchange occurs, suggesting that the regulation of caveolae and caveolins may be pivotal to the net flux of cholesterol between cells and lipoproteins when they are bound to SR-BI.

SR-BII, an isoform of the scavenger receptor BI containing an alternate cytoplasmic tail, mediates lipid transfer between high density lipoprotein and cells.

The scavenger receptor class B, type I (SR-BI), binds high density lipoprotein (HDL) and mediates selective uptake of cholesteryl ester from HDL and HDL-dependent cholesterol efflux from cells. We recently identified a new mRNA variant that differs from the previously characterized form in that the encoded C-terminal cytoplasmic domain is almost completely different. In the present study, we demonstrate that the mRNAs for mouse SR-BI and SR-BII (previously termed SR-BI.2) are the alternatively spliced products of a single gene. The translation products predicted from human, bovine, mouse, hamster, and rat cDNAs exhibit a high degree of sequence similarity within the SR-BII C-terminal domain (62-67% identity when compared with the human sequence), suggesting that this variant is biologically important. SR-BII protein represents approximately 12% of the total immunodetectable SR-BI/II protein in mouse liver. Subcellular fractionation of transfected Chinese hamster ovary cells showed that SR-BII, like SR-BI, is enriched in caveolae, indicating that the altered cytoplasmic tail does not affect targeting of the receptor. SR-BII mediated both selective cellular uptake of cholesteryl ether from HDL as well as HDL-dependent cholesterol efflux from cells, although with approximately 4-fold lower efficiency than SR-BI. In vivo studies using adenoviral vectors showed that SR-BII was relatively less efficient than SR-BI in reducing plasma HDL cholesterol. These studies show that SR-BII, an HDL receptor isoform containing a distinctly different cytoplasmic tail, mediates selective lipid transfer between HDL and cells, but with a lower efficiency than the previously characterized variant.

Characterization of a cytosolic heat-shock protein-caveolin chaperone complex. Involvement in cholesterol trafficking.

Caveolin is a 22-kDa protein that appears to play a critical role in regulating the cholesterol concentration of caveolae. Even though caveolin is thought to be a membrane protein, several reports suggest that this peculiar protein can traffic independently of membrane vesicles. We now present evidence that a cytosolic pool of caveolin is part of a heat-shock protein-immunophilin chaperone complex consisting of caveolin, heat-shock protein 56, cyclophilin 40, cyclophilin A, and cholesterol. Treatment of NIH 3T3 cells with 1 microM cyclosporin A or 100 nM rapamycin disrupted the putative transport complex and prevented rapid (10-20 min) transport of cholesterol to caveolae. The lymphoid cell line, L1210-JF, does not express caveolin, does not form an immunophilin-caveolin complex, and does not transport newly synthesized cholesterol to caveolae. Transfection of caveolin cDNA into L1210-JF cells allowed the assembly of a transport complex identical to that found in NIH 3T3 cells. In addition, newly synthesized cholesterol in transfected cells was rapidly (10-20 min) and specifically transported to caveolae. These data strongly suggest that a caveolin-chaperone complex is a mechanism by which newly synthesized cholesterol is transported from the endoplasmic reticulum through the cytoplasm to caveolae.

Murine SR-BI, a high density lipoprotein receptor that mediates selective lipid uptake, is N-glycosylated and fatty acylated and colocalizes with plasma membrane caveolae.

The class B, type I scavenger receptor, SR-BI, was the first molecularly well defined cell surface high density lipoprotein (HDL) receptor to be described. It mediates transfer of lipid from HDL to cells via selective lipid uptake, a mechanism distinct from receptor-mediated endocytosis via clathrin-coated pits and vesicles. SR-BI is expressed most abundantly in steroidogenic tissues (adrenal gland, ovary), where trophic hormones coordinately regulate its expression with steroidogenesis, and in the liver, where it may participate in reverse cholesterol transport. Here we have used immunochemical methods to study the structure and subcellular localization of murine SR-BI (mSR-BI) expressed either in transfected Chinese hamster ovary cells or in murine adrenocortical Y1-BS1 cells. mSR-BI, an approximately 82-kDa glycoprotein, was initially synthesized with multiple high mannose N-linked oligosaccharide chains, and some, but not all, of these were processed to complex forms during maturation of the protein in the Golgi apparatus. Metabolic labeling with [3H]palmitate and [3H]myristate demonstrated that mSR-BI was fatty acylated, a property shared with CD36, another class B scavenger receptor, and other proteins that concentrate in specialized, cholesterol- and glycolipid-rich plasma membrane microdomains called caveolae. OptiPrep density gradient fractionation of plasma membranes established that mSR-BI copurified with caveolin-1, a constituent of caveolae; and immunofluorescence microscopy demonstrated that mSR-BI colocalized with caveolin-1 in punctate microdomains across the surface of cells and on the edges of cells. Thus, mSR-BI colocalizes with caveolae, and this raises the possibility that the unique properties of these specialized cell surface domains may play a critical role in SR-BI-mediated transfer of lipids between lipoproteins and cells.

Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains.

Results of transgenetic studies argue that the scrapie isoform of the prion protein (PrPSc) interacts with the substrate cellular PrP (PrPC) during conversion into nascent PrPSc. While PrPSc appears to accumulate primarily in lysosomes, caveolae-like domains (CLDs) have been suggested to be the site where PrPC is converted into PrPSc. We report herein that CLDs isolated from scrapie-infected neuroblastoma (ScN2a) cells contain PrPC and PrPSc. After lysis of ScN2a cells in ice-cold Triton X-100, both PrP isoforms and an N-terminally truncated form of PrPC (PrPC-II) were found concentrated in detergent-insoluble complexes resembling CLDs that were isolated by flotation in sucrose gradients. Similar results were obtained when CLDs were purified from plasma membranes by sonication and gradient centrifugation; with this procedure no detergents are used, which minimizes artifacts that might arise from redistribution of proteins among subcellular fractions. The caveolar markers ganglioside GM1 and H-ras were found concentrated in the CLD fractions. When plasma membrane proteins were labeled with the impermeant reagent sulfo-N-hydroxysuccinimide-biotin, both PrPC and PrPSc were found biotinylated in CLD fractions. Similar results on the colocalization of PrPC and PrPSc were obtained when CLDs were isolated from Syrian hamster brains. Our findings demonstrate that both PrPC and PrPSc are present in CLDs and, thus, support the hypothesis that the PrPSc formation occurs within this subcellular compartment.