Steven R. Post

Scavenger receptor-A mediates gp96/GRP94 and calreticulin internalization by antigen-presenting cells

gp96 (GRP94) elicits antigen‐presenting cell (APC) activation and can direct peptides into the cross‐ presentation pathways of APC. These responses arise through interactions of gp96 with Toll‐like (APC activation) and endocytic (cross‐presentation) receptors of APC. Previously, CD91, the α2‐macroglobulin receptor, was identified as the heat shock/chaperone protein receptor of APC. Recent data indicates, however, that inhibition of CD91 ligand binding does not alter gp96 recognition and uptake. Furthermore, CD91 expression is not itself sufficient for gp96 binding and internalization. We now report that scavenger receptor class‐A (SR‐A), a prominent scavenger receptor of macrophages and dendritic cells, serves a primary role in gp96 and calreticulin recognition and internalization. gp96 internalization and peptide re‐presentation are inhibited by the SR‐A inhibitory ligand fucoidin, although fucoidin was without effect on α2‐macroglobulin binding or uptake. Ectopic expression of SR‐A in HEK 293 cells yielded gp96 recognition and uptake activity. In addition, macrophages derived from SR‐A−/− mice were substantially impaired in gp96 binding and uptake. These data identify new roles for SR‐A in the regulation of cellular responses to heat shock proteins.

SREC-I, a Type F Scavenger Receptor, Is an Endocytic Receptor for Calreticulin

Calreticulin and gp96 (GRP94) traffic associated peptides into the major histocompatibility complex class-I cross-presentation pathway of antigen-presenting cells (APCs). Efficient accession of the cross-presentation pathway requires APC receptor-mediated endocytosis of the chaperone/peptide complexes. Previously, scavenger receptor class-A (SRA) was shown to play a substantial role in trafficking gp96 and calreticulin into macrophages, accounting for half of total receptor-mediated uptake. However, the scavenger receptor ligand fucoidin competed the chaperone uptake beyond that accounted for by SRA, indicating that another scavenger receptor(s) may also contribute. Consistent with this hypothesis, we showed that the residual calreticulin uptake into SRA-/- macrophages is competed by the scavenger receptor ligand acetylatedlow density lipoprotein (LDL). We now report that an additional scavenger receptor, SREC-I (scavenger receptor expressed by endothelial cell-I), mediates the endocytosis of calreticulin and gp96. Ectopic expression of SREC-I in Chinese hamster ovary cells yielded chaperone recognition and uptake, and these processes were competed by the inhibitory ligands fucoidin and acetylated (Ac)LDL. Although AcLDL competes for the chaperone interactions with SRA and SREC, we showed that not all of the scavenger receptors, which bind AcLDL, bind calreticulin or gp96. The overexpression of SREC-I in macrophages increased chaperone endocytosis, indicating that SREC-I functions in APCs and that the cytosolic components necessary for the endocytosis of SREC-I and its cargo are present and not limiting in APCs. These data identify a novel class of ligands for SREC-I and provide insight into the mechanisms by which APCs and potentially endothelial cells traffic chaperone/antigen complexes.

Phospholamban-to-SERCA2 ratio controls the force-frequency relationship

The force-frequency relationship (FFR) describes the frequency-dependent potentiation of cardiac contractility. The interaction of the sarcoplasmic reticulum Ca2+-adenosinetriphosphatase (SERCA2) with its inhibitory protein phospholamban (PLB) might be involved in the control of the FFR. The FFR was analyzed in two systems in which the PLB-to-SERCA2 ratio was modulated. Adult rabbit cardiac myocytes were transduced with adenovirus encoding for SERCA2, PLB, and β-galactosidase (control). After 3 days, the relative PLB/SERCA2 values were significantly different between groups (SERCA2, 0.5; control, 1.0; PLB, 4.5). SERCA2 overexpression shortened relaxation by 23% relative to control, whereas PLB prolonged relaxation by 39% and reduced contractility by 47% (0.1 Hz). When the stimulation frequency was increased to 1.5 Hz, myocyte contractility was increased by 30% in control myocytes. PLB-overexpressing myocytes showed an augmented positive FFR (+78%), whereas SERCA2-transduced myocytes displayed a negative FFR (-15%). A more negative FFR was also found in papillary muscles from SERCA2 transgenic mice. These findings demonstrate that the ratio of phospholamban to SERCA2 is an important component in the control of the FFR.

Standardizing Scavenger Receptor Nomenclature

Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a variety of ligands, including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by the U.S. National Institute of Allergy and Infectious Diseases, National Institutes of Health to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of non-self or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. The discussion and nomenclature recommendations described in this report only refer to mammalian scavenger receptors. The purpose of this article is to describe the proposed mammalian nomenclature and classification developed at the workshop and to solicit additional feedback from the broader research community.

ATP activates cAMP production via multiple purinergic receptors in MDCK-D1 epithelial cells. Blockade of an autocrine/paracrine pathway to define receptor preference of an agonist.

Extracellular nucleotides regulate function in many cell types via activation of multiple P2-purinergic receptor subtypes. However, it has been difficult to define which individual subtypes mediate responses to the physiological agonist ATP. We report a novel means to determine this by exploiting the differential activation of an autocrine/paracrine signaling pathway. We used Madin-Darby canine kidney epithelial cells (MDCK-D1) and assessed the regulation of cAMP formation by nucleotides. We found that ATP, 2-methylthio-ATP (MT-ATP) and UTP increase cAMP production. The cyclooxygenase inhibitor indomethacin completely inhibited UTP-stimulated, did not inhibit MT-ATP-stimulated, and only partially blocked ATP-stimulated cAMP formation. In parallel studies, ATP and UTP but not MT-ATP stimulated prostaglandin production. By pretreating cells with indomethacin to eliminate the P2Y2/prostaglandin component of cAMP formation, we could assess the indomethacin-insensitive P2 receptor component. Under these conditions, ATP displayed a ten-fold lower potency for stimulation of cAMP formation compared with untreated cells. These data indicate that ATP preferentially activates P2Y2 relative to other P2 receptors in MDCK-D1 cells (P2Y1 and P2Y11, as shown by reverse transcriptase polymerase chain reaction) and that P2Y2 receptor activation is the principal means by which ATP increases cAMP formation in these cells. Blockade of autocrine/paracrine signaling can aid in dissecting the contribution of multiple receptor subtypes activated by an agonist.

Extracellular ATP and cAMP as Paracrine and Interorgan Regulators of Renal Function P2Y Receptors of MDCK Cells: Epithelial Cell Regulation by Extracellular Nucleotides

1. Madin–Darby canine kidney (MDCK) cells, a well‐ differentiated renal epithelial cell line derived from distal tubule/collecting duct, respond to extracellular nucleotides by altering ion flux and the production of arachidonic acid‐derived products, in particular prostaglandin E2 (PGE2). Our work has defined the receptors and signalling events involved in such responses.

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-AΔ1–55) greatly reduced receptor protein abundance. Retaining the six amino acids proximal to the membrane (SR-AΔ1–49) restored receptor protein abundance. Although SR-AΔ1–49 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-AΔ1–49 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.

Phosphatidylinositol-3-Kinase Regulates Scavenger Receptor Class B Type I Subcellular Localization and Selective Lipid Uptake in Hepatocytes

Objective—The high-density lipoprotein (HDL) receptor scavenger receptor Class B type I (SR-BI) plays a key role in mediating the final step of reverse cholesterol transport. This study examined the possible regulation of hepatic SR-BI by phosphatidylinositol-3-kinase (PI3K), a well known regulator of endocytosis and membrane protein trafficking. Methods and Results—SR-BI–dependent HDL selective cholesterol ester uptake in human HepG2 hepatoma cells was decreased (≈50%) by the PI3K inhibitors wortmannin and LY294002. Insulin increased selective uptake (≈30%), and this increase was blocked by PI3K inhibitors. Changes in SR-BI activity could be accounted for by pronounced changes in the subcellular localization and cell surface expression of SR-BI as determined by HDL cell surface binding, receptor biotinylation studies, and confocal fluorescence microscopy of HepG2 cells expressing green fluorescent protein–tagged SR-BI. Thus, under conditions of PI3K activation by insulin, and to a lesser extent by the SR-BI ligand HDL, cell surface expression of SR-BI was promoted, resulting in increased SR-BI–mediated HDL selective lipid uptake. Conclusion—Our data indicate that PI3K activation stimulates hepatic SR-BI function post-translationally by regulating the subcellular localization of SR-BI in a P13K-dependent manner. Decreased hepatocyte PI3K activity in insulin-resistant states, such as type 2 diabetes, obesity, or metabolic syndrome, may impair reverse cholesterol transport by reducing cell surface expression of SR-BI.

A key role for protein kinase A in homologous desensitization of the beta 2-adrenergic receptor pathway in S49 lymphoma cells.

We have used a [3H]forskolin binding assay to assess Gs-adenylyl cyclase interactions in intact wild-type (WT) and kin− S49 cells under conditions that desensitize the β2-adrenergic receptor (β2-AR) system. This assay provides a measurement of Gαs-adenylyl cyclase interaction that does not rely on the determination of second messenger accumulation or enzyme activity in broken cells. Kin− S49 cells lack protein kinase A (PKA) activity and provide a unique system in which to study the relative importance of this enzyme in β2-AR desensitization. Although both WT and kin− S49 cells display similar kinetics of cAMP accumulation and agonist-induced cell-surface β2-AR loss, we found that these cell types exhibited very different extents of desensitization of forskolin binding following agonist treatment. Specifically, 10 μM isoproterenol (37°C, 30 min) induced the loss of 70% of [3H]forskolin binding sites in WT cells but only 30% in kin− cells. This loss of sites in WT cells displayed a t1/2 of ≈7 min, was agonist concentration-dependent (EC50 ≈ 60 nM), was not mimicked by 8-Br-cAMP, and could be blocked by the PKA inhibitor, H89. The difference between WT and kin− cells in agonist-induced desensitization of the β2-AR pathway was also noted in studies of cAMP accumulation in cells. In addition, preincubation of intact cells with isoproterenol did not inhibit guanine nucleotide-dependent [3H]forskolin binding in permeabilized cells. Overall, data obtained from [3H]forskolin binding assays demonstrate the involvement of PKA in the agonist-dependent uncoupling of β2-AR and Gs; thus we conclude that PKA plays an important role in the homologous desensitization of the β2-AR-Gs-adenylyl cyclase pathway in intact cells.

Inhibition of Phospholipase A2-mediated Arachidonic Acid Release by Cyclic AMP Defines a Negative Feedback Loop for P2Y Receptor Activation in Madin-Darby Canine Kidney D1 Cells

In Madin-Darby canine kidney D1cells extracellular nucleotides activate P2Y receptors that couple to several signal transduction pathways, including stimulation of multiple phospholipases and adenylyl cyclase. For one class of P2Y receptors, P2Y2 receptors, this stimulation of adenylyl cyclase and increase in cAMP occurs via the conversion of phospholipase A2 (PLA2)-generated arachidonic acid (AA) to prostaglandins (e.g. PGE2). These prostaglandins then stimulate adenylyl cyclase activity, presumably via activation of prostanoid receptors. In the current study we show that agents that increase cellular cAMP levels (including PGE2, forskolin, and the β-adrenergic agonist isoproterenol) can inhibit P2Y receptor-promoted AA release. The protein kinase A (PKA) inhibitor H89 blocks this effect, suggesting that this feedback inhibition occurs via activation of PKA. Studies with PGE2indicate that inhibition of AA release is attributable to inhibition of mitogen-activated protein kinase activity and in turn of P2Y receptor stimulated PLA2 activity. Although cAMP/PKA-mediated inhibition occurs for P2Yreceptor-promoted AA release, we did not find such inhibition for epinephrine (α1-adrenergic) or bradykinin-mediated AA release. Taken together, these results indicate that negative feedback regulation via cAMP/PKA-mediated inhibition of mitogen-activated protein kinase occurs for some, but not all, classes of receptors that promote PLA2 activation and AA release. We speculate that receptor-selective feedback inhibition occurs because PLA2activation by different receptors in Madin-Darby canine kidney D1 cells involves the utilization of different signaling components that are differentially sensitive to increases in cAMP or, alternatively, because of compartmentation of signaling components.