Receptor Protein Abundance Research Articles

Class A Scavenger Receptor-mediated Adhesion and Internalization Require Distinct Cytoplasmic Domains

Class A scavenger receptors (SR-A) are transmembrane glycoproteins that mediate both ligand internalization and cell adhesion. Previous studies have identified specific amino acids in the cytoplasmic tail of SR-A that regulate receptor internalization; however, the role of cytoplasmic domains in regulating cell adhesion has not been addressed. To investigate the role of cytoplasmic domains in SR-A-mediated adhesion and to address whether SR-A-mediated adhesion and internalization require distinct cytoplasmic domains, different SR-A constructs were stably expressed in human embryonic kidney (HEK 293) cells. Deleting the entire cytoplasmic tail (SR-A Delta 1-55) greatly reduced receptor protein abundance. Retaining the six amino acids proximal to the membrane (SR-A Delta 1-49) restored receptor protein abundance. Although SR-A Delta 1-49 localized to the cell surface, cells expressing this receptor failed to internalize the ligand acetylated low density lipoprotein. Replacing the cytoplasmic tail of SR-A with that of the transferrin receptor (TfR/SR-A) resulted in retention of the chimeric receptor in the endoplasmic reticulum suggesting a specific role for the membrane-proximal amino acids in trafficking SR-A from the endoplasmic reticulum to the Golgi. Like SR-A expressing cells, cells expressing SR-A Delta 1-49 displayed increased spreading and adhesion, demonstrating that the membrane-proximal amino acids were sufficient for SR-A-mediated cell adhesion. Together, our results indicate a critical role for the membrane-proximal amino acids in SR-A trafficking and demonstrate that SR-A-mediated adhesion and internalization require distinct cytoplasmic domains.

Dopamine-mediated inhibition of renal Na,K-ATPase is reduced by insulin.

Recently we have reported that rosiglitazone treatment of obese Zucker rats reduced plasma insulin and restored the ability of dopamine to inhibit Na,K-ATPase (NKA) in renal proximal tubules. The present study was performed to test the hypothesis that a chronic increase in levels of insulin causes a decrease in expression of the D1 receptor and its uncoupling from G proteins, which may account for the diminished inhibitory effect of dopamine on NKA in obese Zucker rats. We conducted experiments in primary proximal tubule epithelial cells obtained from Sprague-Dawley rat kidneys. These cells at 80% to 90% confluence were pretreated with insulin (100 nmol/L for 24 hours) in growth factor-/serum-free medium. SKF-38393, a D1 receptor agonist, inhibited NKA activity in untreated cells, but the agonist failed to inhibit enzyme activity in insulin-pretreated cells. Basal NKA activity was similar in untreated and insulin-pretreated cells. Measurement of D1 receptors in the plasma membranes revealed that [3H]SCH-23390 binding, a D1 receptor ligand, as well as D1 receptor protein abundance, was significantly reduced in insulin-pretreated cells compared with untreated cells. SKF-38393 (10 micromol/L) elicited significant stimulation of [35S]GTPgammaS binding in the membranes from control cells, suggesting that the D1 receptor-G protein coupling was intact. However, the stimulatory effect of SKF-38393 was absent in membranes from insulin-pretreated cells. We suggest that chronic exposure of cells to insulin causes both the reduction in D1 receptor abundance and its uncoupling from G proteins. These phenomena might account for the diminished inhibitory effect of dopamine on NKA activity in hyperinsulinemic rats.

Transforming growth factor-β1 signaling contributes to Caco-2 cell growth inhibition induced by 1,25(OH)2D3

Growth of Caco-2 and many cancer cells is inhibited by 1,25(OH)(2)D(3). Whereas TGF-beta 1 inhibits normal colonic epithelial cell growth, most human colon cancer-derived cells, including Caco-2 and SW480 cells, are resistant to it. The mechanisms underlying these antiproliferative actions and resistance to TGF-beta growth inhibition are largely unknown. We observed that 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] sensitized Caco-2 and SW480 cells to TGF-beta 1 growth inhibitory effects. Versus 1,25(OH)(2)D(3) alone, the combination of 1,25(OH)(2)D(3) and TGF-beta 1 significantly reduced cell numbers. Also, the amount of active TGF-beta 1 was increased (~4-fold) by this secosteroid in conditioned media from Caco-2 cells. The 1,25(OH)(2)D(3) increased the expression of IGF-II receptors (IGF-IIR), which facilitated activation of latent TGF-beta 1, and was found to activate TGF-beta signaling in Caco-2 cells. By using neutralizing antibodies to human TGF-beta 1, we showed that this cytokine contributes to secosteroid-induced inhibition of Caco-2 cell growth. Also, 1,25(OH)(2)D(3) was found to enhance the type I TGF-beta receptor mRNA and protein abundance in Caco-2 cells. Whereas the 1,25(OH)(2)D(3)-induced sensitization of Caco-2 cells to TGF-beta 1 was IGF-IIR independent, the type I TGF-beta 1 receptor was required for this sensitization. Thus 1,25(OH)(2)D(3) treatment of Caco-2 cells results in activation of latent TGF-beta 1, facilitated by the enhanced expression of IGF-IIR by this secosteroid. Also, 1,25(OH)(2)D(3) sensitized Caco-2 cells to growth inhibitory effects of TGF-beta 1, contributing to the inhibition of Caco-2 cell growth by this secosteroid.

Augmented expression of cardiotrophin-1 in failing human hearts is accompanied by diminished glycoprotein 130 receptor protein abundance.

Cardiotrophin-1 (CT-1), a member of the interleukin-6 superfamily, is a potent inducer of cardiomyocyte hypertrophy that prolongs myocyte survival. Although cardiac CT-1 gene expression is known to be upregulated in some animal models of congestive heart failure, the activation state of the CT-1 system in patients with congestive heart failure is unknown. This study was designed to determine left ventricular expression of CT-1 and its glycoprotein 130 (gp130)/leukemia inhibitory factor receptor complex in human end-stage heart failure due to ischemic and dilated cardiomyopathy. In addition, we investigated the activation state of signal transducer and activator of transcription 3 (STAT3), the downstream effector of gp130 signaling. In the failing left ventricular myocardium, expression levels of CT-1 mRNA and protein were significantly increased by 142% and 68%, respectively, compared with non-failing donor hearts. Immunohistochemistry confirmed the increased expression of CT-1 in cardiac myocytes. Although gp130 gene expression was increased by 91% (P<0.001), gp130 protein abundance was significantly diminished by 34% in the failing myocardium. In contrast, leukemia inhibitory factor receptor and suppressor of cytokine signaling-3 protein concentrations were not changed. In addition, the ratio of activated tyrosine phosphorylated STAT3 to total STAT3 was not significantly altered in failing hearts compared with non-failing controls. Our data suggest that gp130 receptor downregulation balances enhanced CT-1 expression in human heart failure and thereby inhibits excessive activation of the gp130 signaling pathway.

Down-regulation of hepatic high-density lipoprotein receptor, SR-B1, in nephrotic syndrome

Nephrotic syndrome (NS) is a prototype of acquired hypercholesterolemia. Hepatic synthesis and removal of cholesterol play major roles in the regulation of plasma concentration of this sterol. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particles are the primary vehicles for cholesterol transport to the liver. We have recently demonstrated that NS results in acquired hepatic LDL receptor deficiency in rats. This study was undertaken to determine the effect of NS on hepatic expression of the newly discovered, long-sought HDL receptor. Hepatic HDL receptor and apolipoprotein A-I (apo A-I) expressions were studied in rats with puromycin-induced NS. The results were compared with those obtained in placebo-treated, normal controls. The NS group exhibited a marked reduction in hepatic tissue HDL receptor protein abundance when compared with the control group. In contrast, hepatic HDL receptor mRNA abundance in the NS group was similar to that of the control group. As expected, the NS group showed a marked increase in hepatic apo A-I mRNA abundance. The study explored the effect of experimental NS on hepatic HDL receptor expression, and the results revealed a marked down-regulation of HDL receptor in rats with NS. In contrast, hepatic expression of Apo A-I, the principal protein constituent of HDL, was markedly increased in NS rats. The HDL receptor deficiency shown here can potentially limit the efficiency of HDL as the primary vehicle for reverse cholesterol transport in NS.

Hepatic HDL receptor, SR-B1 and Apo A-I expression in chronic renal failure.

Chronic renal failure (CRF) is associated with hypertriglyceridaemia and depressed plasma high-density lipoprotein (HDL)-cholesterol and apolipoprotein A-I (Apo A-I) concentrations. Uraemic hypertriglyceridaemia is due, in part, to lipoprotein lipase and hepatic lipase deficiencies, which are causally linked to excess parathormone (PTH). This study was designed to test the hypothesis that depressed plasma concentration and abnormal composition of HDL in CRF may be due to dysregulation of hepatic expression of Apo A-I and/or the newly discovered HDL receptor. Hepatic Apo A-I and HDL receptor mRNA abundance (Northern blot), and HDL receptor protein mass (Western blot) were determined in CRF rats (5/6 nephrectomy), parathyroidectomized CRF rats (CRF-PTx) and sham-operated controls. The CRF group exhibited normal hepatic HDL receptor mRNA and HDL receptor protein abundance coupled with reduced hepatic Apo A-I mRNA. Hepatic Apo A-I mRNA, HDL receptor mRNA and protein abundance were not affected by PTx. CRF results in the down-regulation of hepatic Apo A-I gene expression, which accounts for the known reduction in plasma Apo A-I concentration. However, CRF does not affect HDL receptor mRNA or protein expression in this model. Neither Apo A-I nor HDL receptor expression were modified by PTx in CRF rats.

Augmented growth response to IGF-1 via increased IRS-1 in Chinese hamster ovary cells expressing kinase-negative insulin receptors.

Although both increased cell growth and impaired insulin signalling have been associated with diabetes, this association has not been investigated. Insulin-like growth factor-1 (IGF-1), a structural and functional analog of insulin, may play a part in the aberrant insulin receptor-mediated signalling observed in diabetes. To investigate the consequence of this impaired signalling on cell proliferation and transformation, we transfected Chinese hamster ovary cells with cDNA encoding a kinase-defective insulin receptor. In these mutant cells, the mitogenic and metabolic effects of insulin were reduced compared with control cells (p < 0.05) and this was due to a dominant negative effect. In contrast, these mutant cells showed a higher mitogenic response to IGF-1 than control cells, although IGF-1 receptor expression was similar in both cell lines. There was no statistically significant difference in mitogenic response, however, to platelet-derived growth factor, basic fibroblast growth factor and heparin-binding epidermal growth factor-like growth factor. Variables of the IGF-1 signalling pathway, including tyrosine phosphorylation of insulin receptor substrate-1 and activation of mitogen-activated protein kinase and phosphatidyl inositol 3 kinase, were also augmented in mutant cells. Insulin receptor substrate-1 message and protein abundance were higher in mutant than in control cells. Moreover, mutant cells had a higher mitogenic potential in low-serum-containing medium, suggesting that these cells have a transformed phenotype. These findings suggest that an impaired insulin signalling may upregulate insulin receptor substrate-1 and that this, in turn, leads to increased IGF-1 signalling, a phenomenon that is possibly associated with increased cell growth in diabetes.

Effect of increased afterload on cardiac lipoprotein lipase and VLDL receptor expression

Fatty acids are a major source of fuel for energy production by myocytes. Lipoprotein lipase (LPL) and very low density lipoprotein (VLDL) receptor are abundantly expressed by the heart and skeletal muscles. LPL and possibly VLDL receptor represent the primary route of access to fatty acids contained in circulating triglyceride-rich lipoproteins. Physical exercise and thyroid hormone, which promote energy consumption, upregulate LPL expression in skeletal muscles. This study tested the hypothesis that increased cardiac workload might modulate myocardial LPL and/or VLDL receptor expressions. Accordingly, cardiac tissue LPL activity, LPL and VLDL receptor proteins and mRNA abundance were studied in Sprague–Dawley rats 4 weeks after induction of severe thoracic aorta constriction or sham operation. Elevation of afterload with thoracic aortic constriction led to a significant cardiomegaly and a marked upregulation of cardiac LPL activity, LPL mRNA and LPL protein abundance, but did not modify VLDL receptor mRNA or protein abundance. Thus, increased cardiac workload in this model results in upregulation of myocardial LPL expression which can enhance fatty acid availability to accommodate the heart’s increased energy requirement.

Expression of calcium channels in adult cardiac myocytes is regulated by calcium.

In addition to playing a significant role in cardiac excitation-contraction coupling, intracellular Ca2+ ([Ca2+]i) can regulate gene expression. While the mechanisms regulating expression of Ca2+ channels are not entirely defined, some evidence exists for Ca2+-dependent regulation. Using an adult ventricular myocyte culture system, we determined the effects of Ca2+ on: (1) abundance of mRNA for L-type Ca2+ channel alpha1 subunit (DHP receptor); (2) amount of DHP receptors; and (3) whole-cell Ca2+ current (ICa). Rat ventricular myocytes were cultured for 1-3 days in serum-free medium containing either normal (1.8 mM) or high (4.8 mM) Ca2+. Exposing myocytes to high Ca2+ rapidly elevated [Ca2+]i as determined by fura-2. Northern blot analysis revealed that culturing cells in high Ca2+ produced 1.5-fold increase in mRNA levels for the DHP receptor. The abundance of DHP receptors, determined by ligand binding, was two-fold greater in myocytes after 3 days in high Ca2+. Moreover, peak ICa was larger in myocytes cultured for 3 days in high Ca2+ (-17.8+/-1.5 pA/pF, n=26) than in control cells (-11.0+/-1.0 pA/pF, n=23). Voltage-dependent activation and inactivation, rates of current decay, as well as percent increases in ICa elicited by Bay K8644 were similar in all groups. Therefore, larger ICa is likely to represent a greater number of functional channels with unchanged kinetics. Our data support the conclusion that transient changes in [Ca2+]i can modulate DHP receptor mRNA and protein abundance, producing a corresponding change in functional Ca2+ channels in adult ventricular myocytes.

Dietary linoleic acid increases and palmitic acid decreases hepatic LDL receptor protein and mRNA abundance in young pigs

The present study was conducted to determine the effects of dietary fatty acids on hepatic LDL receptor (LDLr) protein abundance and mRNA levels. Sixty pigs were randomized into 10 groups and fed corn-soybean meal diets containing three cholesterol levels (0.25%, 0.5%, and 1.0%, w/w) with no added fat, or fats rich (30% of calories) in palmitic acid or linoleic acid. A control group was fed the base diet with no added fat. After 30 days, plasma LDL-cholesterol (LDL-C) levels increased as the dietary cholesterol increased (P < 0.05); however, there was no significant effect of either fatty acid. Dietary fatty acids, however, had distinctly different effects on hepatic LDLr protein (analyzed by ELISA) and mRNA (analyzed by Northern blot) abundance. When pigs consumed diets containing 0.25% cholesterol, linoleic acid increased hepatic LDLr protein 40% whereas palmitic acid reduced it 40% (P < 0.05). These changes in LDLr protein abundance were accompanied by parallel changes in hepatic LDLr mRNA; linoleic acid increased LDLr mRNA 2-fold (P < 0.01), whereas palmitic acid decreased it 60% (P < 0.01). The differential effects of fatty acids on LDLr expression were only observed at 0.25% cholesterol, suggesting that higher intakes of cholesterol have a dominant and repressive effect on regulation of LDLr expression. Cholesterol intake increased hepatic total cholesterol levels (P < 0.01) while dietary fatty acids had no effect on hepatic sterols. In summary, our results indicate that dietary linoleic acid and palmitic acid have markedly different effects on hepatic LDLr protein abundance that are mediated by differential effects on LDLr mRNA and protein levels. Further studies are needed to fully elucidate the molecular mechanisms by which fatty acids regulate LDLr mRNA and protein levels.

Type II TGF(beta) receptor expression in intestinal cell lines and in the intestinal tract.

The recent identification and cloning of mammalian transforming growth factor beta (TGFbeta) receptors permits further analysis of the importance of the TGFbeta family in intestinal biology. Expression of the type II TGFbeta receptor was examined in gastrointestinal cell lines and tissues. The 5.5 kb type II mRNA species was detected in poly-(A) mRNA isolated from the rat small bowel and colon. Northern blot analysis of RNA isolated from epithelial and non-epithelial small intestinal cell fractions showed the majority of receptor mRNA localized in the non-epithelial compartment. Immunohistochemical localization in the small intestine and colon supported the RNA findings; that is, expression was greatest in the lamina propria and muscularis. Staining was also detectable in the epithelium, where it was most prominent in the villus tip cells and absent in crypt cells. These findings mirror expression of TGFbeta in the epithelial compartment. The IEC-6, IPEC and RIE-1 cell lines, all of which are non-transformed, were growth inhibited by TGFbeta and expressed type II receptor mRNA and protein. By contrast, the ras-transfected RIE-1, HT-29, Caco-2 and SW-620 transformed lines were not growth inhibited by TGFbeta and all demonstrated a marked reduction in type II TGFbeta receptor mRNA expression and protein abundance by cross-linking. In conclusion, (i) colocalization of both ligand and receptor establishes the existence of potential autocrine and/or paracrine pathways for TGFbeta in the normal intestine and (ii) down-regulation of the type II TGFbeta receptor occurs in association with cellular transformation and may contribute to intestinal carcinogenesis.