Oligomerization and Endocytosis of the α-Factor Receptor: A Dissertation

α-Factor receptors from Saccharomyces cerevisiae are G-protein-coupled receptors containing seven transmembrane segments. The ability of α-factor receptors to form oligomeric complexes with each other and with other proteins was investigated. Both in vivo and in vitroevidence was obtained that suggests homo-oligomerization of receptors in the plasma membrane. When the membranes from cells coexpressing two differentially-tagged receptors were solubilized with detergent and subjected to immunoprecipitation, the antibodies specific for either epitope tag resulted in precipitation of both tagged species. Treatment of cultures with α-factor had little effect on the extent of oligomerization as judged by the sedimentation behavior of the receptor complexes and by the efficiency of coimmunoprecipitation. The ability of receptor complexes to undergo ligand-mediated endocytosis was evaluated by using membrane fractionation and fluorescence microscopy. Mutant receptors that fail to bind α-factor (Ste2-S184R) or lack the endocytosis signal (Ste2-T326) became competent for ligand-mediated endocytosis when they were expressed in cells containing wild-type receptors. Coimmunoprecipitation experiments indicated that the C-terminal cytoplasmic domain and intermolecular disulfide bonds were unnecessary for oligomer formation. Therefore, α-factor receptors form homo-oligomers and that these complexes are subject to ligand-mediated endocytosis. A crosslinking and immunoprecipitation strategy was used to capture and characterize the transient complexes that contain the α-factor receptor Ste2. Tagged receptors were crosslinked to form at least three high molecular weight complexes and the complexes were immunoprecipitated with antibodies against the tag. Western blotting analysis of the precipitated material revealed the presence of β and γ subunits of the heterotrimeric G protein, Ste4 and Stel8. Similar results were obtained when the cultures had been treated with α-factor prior to analysis. A truncated receptor missing most of the cytoplasmic C-terminal tail was also active in binding Ste4. Overall, these results constitute the first biochemical evidence for a physical association between the α-factor receptor and its cognate G-protein. Endocytic signals in the C-terminal tail (residues 297-431) of the α-factor receptor were analyzed. One signaling element, SINNDAKSS, (residues 331-339) is known to be sufficient (but not necessary) for endocytosis. Internal deletions of the STE2 gene were constructed that remove sequences encoding SINNDAKSS and selected regions of the C-terminal tail. Strains containing these alelles were then assayed for endocytosis in the presence and absence of α-factor. Residues from 360 to 431 were sufficient to mediate both constitutive and ligand-mediated endocytosis of the receptor even though 63 residues including the SINNDAKSS motif had been removed. Structural features of this region that were investigated further were the highly-ubiquitinated Lys374, the neighboring Lys387, and the GPFAD motif (residues 392-396). Lys374 and Lys387 were unnecessary for the element to promote exit from the plasma membrane; however, Lys374 may play some role in intracellular trafficking. The GPFAD motif was not sufficient to promote endocytosis, since the residues 360-399 provided no detectable endocytic activity. Overall, these results suggest that a new region in the C-terminal of the α-factor receptor, redundant with the SINNDAKSS motif, is sufficient to mediate the constitutive endocytosis as well as the ligand-mediated endocytosis of the receptor

Excess cholesterol induces mouse egg activation and may cause female infertility

The HDL receptor scavenger receptor, class B type I (SR-BI) controls the structure and fate of plasma HDL. Female SR-BI KO mice are infertile, apparently because of their abnormal cholesterol-enriched HDL particles. We examined the growth and meiotic progression of SR-BI KO oocytes and found that they underwent normal germinal vesicle breakdown; however, SR-BI KO eggs, which had accumulated excess cholesterol in vivo, spontaneously activated, and they escaped metaphase II (MII) arrest and progressed to pronuclear, MIII, and anaphase/telophase III stages. Eggs from fertile WT mice were activated when loaded in vitro with excess cholesterol by a cholesterol/methyl-β-cyclodextrin complex, phenocopying SR-BI KO oocytes. In vitro cholesterol loading of eggs induced reduction in maturation promoting factor and MAPK activities, elevation of intracellular calcium, extrusion of a second polar body, and progression to meiotic stages beyond MII. These results suggest that the infertility of SR-BI KO females is caused, at least in part, by excess cholesterol in eggs inducing premature activation and that cholesterol can activate WT mouse eggs to escape from MII arrest. Analysis of SR-BI KO female infertility raises the possibility that abnormalities in cholesterol metabolism might underlie some cases of human female infertility of unknown etiology.National Institutes of Health (U.S.)National Institutes of Health (U.S.) (Pre-doctoral Training Grant T32GM007287)Massachusetts Institute of Technology (International Science and Technology Initiatives Chile Cooperative Grant

A Love-Hate Relationship: Cholesterol in Health and Disease

In this class, we will examine cholesterol's role in the cell and in the body as a whole, from its function as a structural component of the membrane to its function in signaling. We will discuss mechanisms of cholesterol sensing, mechanisms of feedback regulation in cells, cholesterol in the brain, cholesterol in the circulation, 'good cholesterol' and 'bad cholesterol,' cholesterol-related human disorders, and the drugs that deal with some of these disorders. This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching

Homo-oligomeric complexes of the yeast alpha-factor pheromone receptor are functional units of endocytosis

alpha-Factor receptors from Saccharomyces cerevisiae are G-protein-coupled receptors containing seven transmembrane segments. Receptors solubilized with the detergent n-dodecyl beta-D-maltoside were found to sediment as a single 8S species in glycerol density gradients. When the membranes from cells coexpressing two differentially tagged receptors were solubilized with detergent and subjected to immunoprecipitation, we found that the antibodies specific for either epitope tag resulted in precipitation of both tagged species. Coprecipitation was not a consequence of incomplete detergent extraction because the abundant plasma membrane protein Pma1 did not coprecipitate with the receptors. Moreover, the receptor complexes were present prior to detergent extraction because coimmunoprecipitation was not observed when cells expressing the single tagged species were mixed prior to membrane preparation. Treatment of cultures with alpha-factor had little effect on the extent of oligomerization as judged by the sedimentation behavior of the receptor complexes and by the efficiency of coimmunoprecipitation. The ability of receptor complexes to undergo ligand-mediated endocytosis was evaluated by using membrane fractionation and fluorescence microscopy. Mutant receptors that fail to bind alpha-factor (Ste2-S184R) or lack the endocytosis signal (Ste2-T326) became competent for ligand-mediated endocytosis when they were expressed in cells containing wild-type receptors. Coimmunoprecipitation experiments indicated that the C-terminal cytoplasmic domain and intermolecular disulfide bonds were unnecessary for oligomer formation. We conclude that alpha-factor receptors form homo-oligomers and that these complexes are subject to ligand-mediated endocytosis. Furthermore, we show for the first time that unoccupied receptors participate in these endocytosis-competent complexes

Loss of PDZK1 Causes Coronary Artery Occlusion and Myocardial Infarction in Paigen Diet-Fed Apolipoprotein E Deficient Mice

Background: PDZK1 is a four PDZ-domain containing protein that binds to the carboxy terminus of the HDL receptor, scavenger receptor class B type I (SR-BI), and regulates its expression, localization and function in a tissue-specific manner. PDZK1 knockout (KO) mice are characterized by a marked reduction of SR-BI protein expression (~95%) in the liver (lesser or no reduction in other organs) with a concomitant 1.7 fold increase in plasma cholesterol. PDZK1 has been shown to be atheroprotective using the high fat/high cholesterol (‘Western’) diet-fed murine apolipoprotein E (apoE) KO model of atherosclerosis, presumably because of its role in promoting reverse cholesterol transport via SR-BI. Principal Findings: Here, we have examined the effects of PDZK1 deficiency in apoE KO mice fed with the atherogenic ‘Paigen’ diet for three months. Relative to apoE KO, PDZK1/apoE double KO (dKO) mice showed increased plasma lipids (33% increase in total cholesterol; 49 % increase in unesterified cholesterol; and 36% increase in phospholipids) and a 26% increase in aortic root lesions. Compared to apoE KO, dKO mice exhibited substantial occlusive coronary artery disease: 375% increase in severe occlusions. Myocardial infarctions, not observed in apoE KO mice (although occasional minimal fibrosis was noted), were seen in 7 of 8 dKO mice, resulting in 12 times greater area of fibrosis in dKO cardiac muscle. Conclusions: These results show that Paigen-diet fed PDZK1/apoE dKO mice represent a new animal model useful for studying coronary heart disease and suggest that PDZK1 may represent a valuable target for therapeutic intervention.National Institutes of Health (Grants HL-52212, HL66105, and HL077780

Effects of loss of PDZK1 on coronary atherosclerosis and cardiac fibrosis in apoE KO mice.

<p>Hearts were harvested from Paigen diet-fed mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008103#s2" target="_blank">Methods</a> (n = 8 per genotype). Left panels: A–B: representative cross-sections of Oil red O-stained (A–B) or trichrome-stained (C) myocardial coronary arterioles, showing unremarkable arterioles in apoE KO (A) and totally occluded arterioles in PDZK1/apoE dKO (B–C) mice (magnification, ×100). The Oil red O stain shows that the coronary arteriole is occluded almost exclusively by lipid-rich lesions (B), while the trichrome stain shows that the arteriole is surrounded by fibrosis in an area of myocardial infarction in a PDZK1/apoE dKO mouse (C). D–E: trichrome stained sections of hearts showing areas of infarction/fibrosis stained blue in PDZK1/apoE dKO (E), while they are absent in apoE KO (D) mice (magnification, ×10). Right top panel: quantification of coronary artery occlusions in apoE KO and PDZK1/apoE dKO mice. Statistically significant differences by ANOVA Tukey posthoc test comparing the two genotypes within a given group are indicated as: P<0.001. Right bottom panel: quantification of cardiac fibrosis in apoE KO and PDZK1/apoE dKO mice. Unpaired Student's t-test was used to determine statistical significance.</p

Plasma lipid levels and body weights of apoE KO and PDZK1/apoE dKO mice.

<p>Four month old animals were fasted for 4 hours prior to sample collection. Values are represented as mean±standard error from 8–25 animals (mean 20 animals) per group. Statistical significance was determined by pairwise comparisons of each value from PDZK1/apoE dKO mice with apoE KO controls by using unpaired Student's t test. The abbreviations and units used are TC (total plasma cholesterol, mg/dL), UC (plasma unesterified cholesterol, mg/dL), UC:TC (plasma unesterified cholesterol to total plasma cholesterol ratio), PL (plasma phospholipids, mg/dL), TG (plasma triglycerides, mg/dL) and Wt (body weights, g).</p>a, b, c, e<p>P<0.01</p>d<p>P<0.05</p

Immunoblot analysis of hepatic SR-BI expression.

<p>Mice with the indicated genotypes were fed a high fat/high cholesterol/cholate-containing “Paigen” diet for three months. Livers were harvested and subjected to immunoblotting using anti-SR-BI and anti-actin (loading control) antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008103#s2" target="_blank">Materials and Methods</a>.</p

Lipoprotein cholesterol profiles from apoE KO and PDZK1/apoE dKO mice.

<p>Plasma harvested from individual mice fed a Paigen diet for three months was size fractionated using FPLC, and the total cholesterol contents of the fractions (mg/dL plasma) were determined by enzymatic assay. Profiles averaged from 2 independent experiments for each genotype, each composed of pooled plasma from six apoE KO (open circles) and six PDZK1/apoE dKO (filled circles) mice per experiment are shown. Approximate elution positions of human VLDL, IDL/LDL and HDL are indicated.</p