Richard A. F. Dixon

Structural basis of beta-adrenergic receptor function.

Receptors that mediate their actions by stimulating guanine nucleotide binding regulatory proteins (G proteins) share structural as well as functional similarities. The structural motif characteristic of receptors of this class includes seven hydrophobic putative transmembrane domains linked by hydrophilic loops. Genetic analysis of the β‐adrenergic receptor (βAR) revealed that the ligand binding domain of this receptor, like that of rhodopsin, involves residues within the hydrophobic core of the protein. On the basis of these studies, a model for ligand binding to the receptor has been developed in which the amino group of an agonist or antagonist is anchored to the receptor through the carboxylate side chain of Asp113 in the third transmembrane helix. Other interactions between specific residues of the receptor and functional groups on the ligand have also been proposed. The interaction between the βAR and the G protein Gs has been shown to involve an intracellular region that is postulated to form an amphi‐philic α helix. This region of the βAR is also critical for sequestration, which accompanies agonist‐mediated desensitization, to occur. Structural similarities among G protein‐linked receptors suggest that the information gained from the genetic analysis of the βAR should help define functionally important regions of other receptors of this class.— Strader, C. D.; Sigal, I. S.; Dixon, R. A. F. Structural basis of β‐adrenergic receptor function. FASEB J. 3: 1825‐1832; 1989.

Molecular cloning and primary structure of human 15-lipoxygenase

A full-length cDNA encoding 15-lipoxygenase has been isolated from a human reticulocyte cDNA library. The predicted primary structure of the enzyme exhibits a sequence similarity of 61% and 45% with human 5-lipoxygenase and the soybean lipoxygenase isoenzyme I, respectively. When all three lipoxygenases are aligned, there are two distinct regions of significant sequence identity including a cluster of five histidine residues conserved in all three lipoxygenases. Because histidines can serve as ligands for the enzymatically active iron, this region may be critical to enzymatic function. These results provide a basis for exploring functional domains of lipoxygenases.

The carboxyl terminus of the hamster β-adrenergic receptor expressed in mouse L cells is not required for receptor sequestration

The structural basis for agonist-mediated sequestration and desensitization of the beta-adrenergic receptor (beta AR) was examined by oligonucleotide-directed mutagenesis of the hamster beta AR gene and expression of the mutant genes in mouse L cells. Treatment of these cells with the agonist isoproterenol corresponded to a desensitization of beta AR activity. A mutant receptor that bound agonist but did not couple to adenylate cyclase showed a dramatically reduced sequestration response to agonist stimulation. In contrast, beta AR mutants in which the C-terminus was truncated and/or in which two regions that have been proposed as phosphorylation substrates for cAMP-dependent protein kinase were removed showed normal sequestration responses. These results demonstrate that agonist-mediated sequestration of the beta AR can occur in the absence of the C-terminus of the protein and reveal a strong correlation between effective coupling to Gs and sequestration.

Affinity purification of the HIV-1 protease

An inhibitor of the HIV-1 protease has been employed in the generation of a resin which allows the rapid purification of this enzyme. A peptide substrate analogue, H2N-Ser-Gln-Asn-(Phe-psi[CH2N]-Pro)-Ile-Val-Gln-OH, was coupled to agarose resin. The HIV-1 protease was expressed in E. coli and the supernatant from lysed cells was passed through the affinity resin. Active HIV-1 protease was then eluted with a buffer change to pH 10 and 2 M NaCl. Final purification to a homogeneous preparation, capable of crystallization, was achieved with hydrophobic interaction chromatography. Solutions containing HIV-1 protease bound to competitive inhibitors do not bind to the column.

Ventricular atrial natriuretic factor in the cardiomyopathic hamster model of congestive heart failure.

Cardiac atria are thought to be the principle source of plasma atrial natriuretic factor (ANF), a potent natriuretic and diuretic peptide. Whether other ANF production sites are recruited in disease states exhibiting elevated plasma ANF levels is not known. Accordingly, in the cardiomyopathic hamster, an animal model of congestive heart failure with high circulating levels of ANF, contribution of ventricular tissue to total cardiac ANF production and storage was investigated. Measurements were made of immunoreactive ANF in plasma and in atrial and ventricular extracts as well as ANF mRNA levels in the atria and ventricles from normal and cardiomyopathic golden Syrian hamsters. Plasma ANF levels were higher in cardiomyopathic than in control animals. The atrial concentration of ANF (per milligram atrial weight) was 50% and 83% lower hi moderate and severe congestive heart failure, respectively, when compared with controls, while atrial ANF mRNA content of cardiomyopathic hamsters was not significantly different from normal hamsters. The ventricular concentration of ANF was 3 times and 7 times higher in animals in moderate and severe heart failure when compared with controls. In severe heart failure, ventricular ANF accounted for 23% of total cardiac stores of ANF. Ventricular ANF mRNA levels were 7 tunes and 13 times higher in hamsters in moderate and severe heart failure as compared with control animals. Therefore, significant increases in both ANF content and ANF mRNA in ventricles of hamsters in moderate to severe heart failure suggest that the ventricle could be an important source of ANF in congestive heart failure.

Integrin α2β1 mediates tyrosine phosphorylation of vascular endothelial cadherin induced by invasive breast cancer cells.

Background: The disruption of endothelial barrier function by tumor cells was studied. Results: The attachment of tumor cells to endothelial cells leads to the disorganization of endothelial adherens junction. Conclusion: Interaction of tumor cells with endothelial cells alters endothelial signaling and facilitates cancer cell diapedesis. Significance: This study introduces new therapeutic targets for treating metastatic breast cancer. The molecular mechanisms that regulate the endothelial response during transendothelial migration (TEM) of invasive cancer cells remain elusive. Tyrosine phosphorylation of vascular endothelial cadherin (VE-cad) has been implicated in the disruption of endothelial cell adherens junctions and in the diapedesis of metastatic cancer cells. We sought to determine the signaling mechanisms underlying the disruption of endothelial adherens junctions after the attachment of invasive breast cancer cells. Attachment of invasive breast cancer cells (MDA-MB-231) to human umbilical vein endothelial cells induced tyrosine phosphorylation of VE-cad, dissociation of β-catenin from VE-cad, and retraction of endothelial cells. Breast cancer cell-induced tyrosine phosphorylation of VE-cad was mediated by activation of the H-Ras/Raf/MEK/ERK signaling cascade and depended on the phosphorylation of endothelial myosin light chain (MLC). The inhibition of H-Ras or MLC in endothelial cells inhibited TEM of MDA-MB-231 cells. VE-cad tyrosine phosphorylation in endothelial cells induced by the attachment of MDA-MB-231 cells was mediated by MDA-MB-231 α2β1 integrin. Compared with highly invasive MDA-MB-231 breast cancer cells, weakly invasive MCF-7 breast cancer cells expressed lower levels of α2β1 integrin. TEM of MCF-7 as well as induction of VE-cad tyrosine phosphorylation and dissociation of β-catenin from the VE-cad complex by MCF-7 cells were lower than in MDA-MB-231 cells. These processes were restored when MCF-7 cells were treated with β1-activating antibody. Moreover, the response of endothelial cells to the attachment of prostatic (PC-3) and ovarian (SKOV3) invasive cancer cells resembled the response to MDA-MB-231 cells. Our study showed that the MDA-MB-231 cell-induced disruption of endothelial adherens junction integrity is triggered by MDA-MB-231 cell α2β1 integrin and is mediated by H-Ras/MLC-induced tyrosine phosphorylation of VE-cad.

Electronegative Low-Density Lipoprotein Increases C-Reactive Protein Expression in Vascular Endothelial Cells through the LOX-1 Receptor

Objectives Increased plasma C-reactive protein (CRP) levels are associated with the occurrence and severity of acute coronary syndrome. We investigated whether CRP can be generated in vascular endothelial cells (ECs) after exposure to the most electronegative subfraction of low-density lipoprotein (LDL), L5, which is atherogenic to ECs. Because L5 and CRP are both ligands for the lectin-like oxidized LDL receptor-1 (LOX-1), we also examined the role of LOX-1. Methods and Results Plasma LDL samples isolated from asymptomatic hypercholesterolemic (LDL cholesterol [LDL-C] levels, 154.6±20 mg/dL; nu200a=u200a7) patients and normocholesterolemic (LDL-C levels, 86.1±21 mg/dL; P<0.001; nu200a=u200a7) control individuals were chromatographically resolved into 5 subfractions, L1-L5. The L5 percentage (L5%) and the plasma L5 concentration ([L5] u200a=u200a L5% × LDL-C) in the patient and control groups were 8.1±2% vs. 2.3±1% (P<0.001) and 12.6±4 mg/dL vs. 1.9±1 mg/dL (P<0.001), respectively. In hypercholesterolemic patients treated with atorvastatin for 6 months (10 mg/day), [L5] decreased from 12.6±4 mg/dL to 4.5±1.1 mg/dL (Pu200a=u200a0.011; nu200a=u200a5), whereas both [L5] and L5% returned to baseline levels in 2 noncompliant patients 3 months after discontinuation. In cultured human aortic ECs (HAECs), L5 upregulated CRP expression in a dose- and time-dependent manner up to 2.5-fold (P<0.01), whereas the least electronegative subfraction, L1, had no effect. DiI-labeled L1, internalized through the LDL receptor, became visible inside HAECs within 30 seconds. In contrast, DiI-labeled L5, internalized through LOX-1, became apparent after 5 minutes. L5-induced CRP expression manifested at 30 minutes and was attenuated by neutralizing LOX-1. After 30 minutes, L5 but not L1 induced reactive oxygen species (ROS) production. Both L5-induced ROS and CRP production were attenuated by ROS inhibitor N-acetyl cysteine. Conclusions Our results suggest that CRP, L5, and LOX-1 form a cyclic mechanism in atherogenesis and that reducing plasma L5 levels with atorvastatin disrupts the vascular toxicity of L5.

Chapter 23. Structure and Function of G-Protein Coupled Receptors

Publisher Summary The receptors that interact with G-proteins share a variety of structural and functional similarities. In particular, there appears to be a common motif for the ligand binding domains of the receptors that involves critical residues within the transmembrane regions. This chapter suggests a role of amphiphilic helices in activation of G-proteins and that the role may provide a target for modulating receptor G-protein interactions independent of the ligand binding site of the receptor. The chapter discusses the role of G-proteins, arrestin, and phosphorylation in receptor desensitization. Many hormones and cellular mediators exert their influence on target cells through interactions with cell surface receptors. The classes of receptors include the growth factor receptors, the transporter receptors, ion channels, and receptors linked to second messenger production through guanine nucleotide binding regulatory proteins. The chapter describes the components involved in signal transduction mediated by G-protein coupled receptors, with each of the components located relative to the membrane bilayer. The first step in this signaling pathway is the interaction of an extracellular message with a receptor that spans the membrane. Once bound to an agonist the ligand receptor complex becomes activated. Antagonist ligands do not activate the receptor. The details of receptor activation are unknown, but probably involve conformational changes within the receptor. The first G-protein linked and adrenergic hormone receptor genes to be cloned were for the β 1 and β 2 adrenergic receptors from Turkey and Hamster, respectively. The predicted amino acid sequences for the seproteins were found to share significant sequence similarity. A combination of the biochemical and genetic analysis has allowed the identification of the ligand binding site, sites of G-protein interaction, and regulatory sites on several of the G-protein linked receptors. By analogy with rhodopsin, it has been proposed that the ligand binding sites of other G-protein linked receptors will also involve the transmembrane helices.

Myosin light chain phosphorylation facilitates monocyte transendothelial migration by dissociating endothelial adherens junctions

AIMSnTransendothelial migration (TEM) of monocytes is a crucial step in inflammatory processes such as atherogenesis. Tyrosine phosphorylation of vascular endothelial cadherin (VE-cad) has been implicated in the dissociation of adherens junctions and the increased paracellular permeability of endothelial cells (ECs) that occur during TEM of monocytes. However, the underlying molecular mechanism has not been determined. We tested the hypothesis that the phosphorylation of myosin light chain (MLC) in ECs is crucial for the dissociation of adherens junctions during TEM of monocytes.nnnMETHODS AND RESULTSnUsing a combination of biochemical and cellular techniques, we provide evidence for the signal transduction pathways that regulate tyrosine phosphorylation of VE-cad in ECs after the attachment of monocytes. Our findings indicate that after interaction of integrins on THP-1 cells with adhesion molecules on ECs, the induction of the HRasRafMEKERK signalling cascade leads to the phosphorylation of MLC. This results in the recruitment of Src to the VE-cad complex and tyrosine phosphorylation of VE-cad, which leads to dissociation of β-catenin from the VE-cad complex, formation of gaps between ECs, and enhancement of THP-1 cell TEM.nnnCONCLUSIONnOur studies suggest that monocyte-induced phosphorylation of MLC in ECs enhances TEM of monocytes through dissociation of EC adherens junctions.

Treatment of hind limb ischemia using angiogenic peptide nanofibers

For a proangiogenic therapy to be successful, it must promote the development of mature vasculature for rapid reperfusion of ischemic tissue. Whole growth factor, stem cell, and gene therapies have yet to achieve the clinical success needed to become FDA-approved revascularization therapies. Herein, we characterize a biodegradable peptide-based scaffold engineered to mimic VEGF and self-assemble into a nanofibrous, thixotropic hydrogel, SLanc. We found that this injectable hydrogel was rapidly infiltrated by host cells and could be degraded while promoting the generation of neovessels. In mice with induced hind limb ischemia, this synthetic peptide scaffold promoted angiogenesis and ischemic tissue recovery, as shown by Doppler-quantified limb perfusion and a treadmill endurance test. Thirteen-month-old mice showed significant recovery within 7 days of treatment. Biodistribution studies in healthy mice showed that the hydrogel is safe when administered intramuscularly, subcutaneously, or intravenously. These preclinical studies help establish the efficacy of this treatment for peripheral artery disease due to diminished microvascular perfusion, a necessary step before clinical translation. This peptide-based approach eliminates the need for cell transplantation or viral gene transfection (therapies currently being assessed in clinical trials) and could be a more effective regenerative medicine approach to microvascular tissue engineering.