Cellular and Molecular Mechanisms of Inflammation (eBook)
266 Seiten
Elsevier Science (Verlag)
978-1-4831-9151-5 (ISBN)
Receptors of Inflammatory Cells: Structure-Function Relationships is the first in a new serial on Cellular and Molecular Mechanisms of Inflammation. The purpose of this serial is to bring together the latest knowledge in various areas of research in this actively developing field around a topical focus. These volumes are not intended to present comprehensive reviews. Rather, each contribution is meant to be a status report from laboratories actively working in an area. This volume presents an analysis of the structure-function relationships of receptors. It is clear that the structure of receptors provides the initial guidance for numerous functions of each cell in the organism. Through an analysis of the submolecular features of the receptors that are responsible for the initiation of activity of diverse biochemical pathways within the cells, a molecular understanding of the all important initial, guiding events of cell functions will emerge. In the broad sense of cells involved in inflammation, this includes mitogenesis, gene transcription, generation of lipid metabolites and oxidants, clearance of molecules from the surrounding medium, and release of granular constituents from cytoplasmic vesicles into the external medium, among others. The contents of this first volume will serve as a foundation for the subject of the second volume, which is signal transduction. Four additional volumes are in preparation, including Endothelial Leukocyte-Adhesion Molecules, Leukocyte Adhesive Mechanisms in Inflammation and Immunity, a second volume on Signal Transduction, and Stimulation of Inflammatory Cells.
Genetic Analysis of β-Adrenergic Receptor Structure and Function
Catherine D. Strader, Department of Molecular, Pharmacology and Biochemistry, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065
Richard A.F. Dixon, Department of Molecular Biology, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486
Publisher Summary
This chapter presents a genetic analysis of β-adrenergic receptor structure. A wide variety of hormone and neurotransmitter receptors stimulate their cellular effects through coupling to guanine nucleotide-binding regulatory proteins (G proteins). The binding of agonist ligands to these receptors results in the formation of a high-affinity complex between the hormone, the receptor, and the GDP-bound form of the heterotrimeric G protein. A formation of this ternary complex reduces the affinity of the G protein for GDP, causing the release of GDP from the guanine nucleotide-binding site of the G protein and allowing GTP to bind. The GTP-bound form of the G protein is the activated form: the GTP-Gα subunit separates from Gβγ and the receptor to activate intracellular effector enzymes. In the absence of an association with the G protein, the affinity of the receptor for the agonist is reduced and the hormone-receptor complex dissociates. The primary structures of several of the G protein-coupled receptors can be determined by cloning and sequence analysis. In situ hybridization experiments can be used to establish the tissue distribution of the newly discovered receptor subtypes, and exogenous expression in mammalian cells can be used to determine their preferred signal transduction pathways and pharmacological profiles. Thus, the availability of cells expressing pure receptor subtypes should enhance the definition of specific therapeutic targets and the development of more selective pharmaceutical agents that act exclusively at a single receptor subtype.
Introduction
A wide variety of hormone and neurotransmitter receptors stimulate their cellular effects through coupling to guanine nucleotide-binding regulatory proteins (G proteins). The transmembrane signaling pathway shared by this class of receptors has been analyzed in detail in several laboratories (1). Briefly, the binding of agonist ligands to these receptors results in the formation of a high-affinity complex between the hormone, the receptor, and the GDP-bound form of the heterotrimeric G protein. Formation of this ternary complex reduces the affinity of the G protein for GDP, causing the release of GDP from the guanine nucleotide-binding site of the G protein, and allowing GTP to bind. The GTP-bound form of the G protein is the activated form: the GTP-Gα subunit separates from Gβγ and the receptor to activate intracellular effector enzymes. In the absence of association with the G protein, the affinity of the receptor for the agonist is reduced and the hormone–receptor complex dissociates. To date, at least nine Gα subunits have been cloned, and their specific functions are being determined by reconstitution studies. Effector systems identified as being stimulated or inhibited by G proteins include adenylyl cyclase, guanylyl cyclase, K+ and Ca2+ channels, phospholipases C and A2, as well as a number of transmembrane ion exchange systems.
The primary structures of several of the G protein-coupled receptors have recently been determined by cloning and sequence analysis, revealing considerable structural homology among these proteins. To date, receptors of this class that have been cloned include the visual opsins, receptors for small biogenic amines (α1-,α2-,β1-,β2-, and β3-adrenergic; D2 dopaminergic; serotonin-1a, -1c, and -2; muscarinic M1–M5), receptors for small peptides (neurokinins 1, 2, and 3; angiotensin), and receptors for larger protein hormones (lutropin–choriogonadotropin; thyrotropin) (2–20). In addition, through the use of polymerize chain reaction (PCR) technology, many other putative G protein-coupled receptors have been cloned, with their specific identities not yet established (21).
One unexpected benefit of the cloning of G protein-coupled receptors has been the discovery of new subtypes of receptors through cross-hybridization with receptor DNA. In many cases, the existence of these receptor subtypes had not been demonstrated by classical pharmacological techniques because of the absence of a tissue source for the pure receptor subtype. Thus, five subtypes of the muscarinic receptor (14), two subtypes of the α2-receptor (4, 5), and an additional β3 β-adrenergic receptor (9) have been identified by the cloning of their genes. In situ hybridization experiments can be used to establish the tissue distribution of the newly discovered receptor subtypes, and exogenous expression in mammalian cells can be used to determine their preferred signal transduction pathways and pharmacological profiles. Thus, the availability of cells expressing pure receptor subtypes should enhance the definition of specific therapeutic targets and the development of more selective pharmaceutical agents that act exclusively at a single receptor subtype.
Receptor Structure
Current efforts in several laboratories are directed toward determining the structure/function relationships for this class of related receptors.
An understanding of the structural domains of the receptors that are responsible for specific functions, such as ligand binding or G protein coupling, should enhance our ability to design agonists or antagonists for a particular receptor. The structural motif shared by the family of G protein-coupled receptors includes seven stretches of hydrophobic amino acids, 20–25 residues in length, which are presumed to form membrane-spanning α-helices. These putative transmembrane helices are connected by hydrophilic loops of varying lengths, predicted to be exposed alternatingly extracellularly and intracellularly. Primary sequence conservation among different receptors ranges from approximately 25 to 80%, with the majority of sequence homology contained within the putative transmembrane domains of the proteins. The N-terminal domains of all of these receptors contain potential sites of relinked glycosylation, suggesting that these regions are extracellular, and leading to the predicted topology which is shown for the β-adrenergic receptor (βAR) in Fig. 1. This topology would dictate that the C-terminus of the protein be exposed to the cytoplasm, in agreement with the observation that this region of many of the receptors contains several potential sites for phosphorylation by intracellular kinases. The intracellular exposure of the hydrophilic loop connecting hydrophobic helices 5 and 6 of the βAR has been demonstrated directly by immunofluorescence using specific antipeptide antibodies (22). In addition, a recent immunofluorescence study by Wang and co-workers provides evidence for the transmembrane distribution of the βAR shown in Fig. 1 (23).
FIGURE 1 Model for the transmembrane disposition of the βAR. The single-letter code is used to designate amino acid residues. The receptor is shown in its proposed transmembrane orientation (see text) with the top of the diagram being extracellular. Residues that could be deleted without affecting ligand binding are shown as squares; highly conserved Cys residues are shown as hexagons. The two sites of N-linked glycosylation are designated with solid squares. The proposed highly conserved disulfide bond linking extracellular loops 2 and 3 is shown. Bold circles show the positions of D113, S204, and S207 (see text). Cylinders encircle the proposed amphipathic α-helices which are required for G protein coupling. [Reprinted with permission from Strader, C. D. et al. American Journal of Cell and Molecular Biology 1, 81–86 (1989).]
This model for the transmembrane disposition and folding of G protein-coupled receptors was originally developed for rhodopsin, and is based on the similarities in predicted secondary structure that were noted between rhodopsin and bacteriorhodopsin (which is not coupled to G proteins) (24). However, all of the G protein-coupled receptors that have been cloned share a similar distribution of hydrophobic and hydrophilic amino acids, leading to the adoption of this structure as a working model for all of these receptors. Currently lacking for any of the receptors is direct biophysical evidence for the existence of any of the predicted secondary structural features or any information about the packing of the putative transmembrane helices in the membrane. None of these receptors has yet been crystallized for X-ray diffraction studies, and the quantities of protein that have been available to date have precluded even spectroscopic analysis of any of the proteins except for rhodopsin. The development of high-level expression systems for the cloned receptors should allow direct biophysical analysis of these proteins in the future. In the interim, site-directed mutagenesis and proteolysis experiments on several of these receptors are providing biochemical and pharmacological evidence with which to refine the current model for the structure of...
Erscheint lt. Verlag | 22.10.2013 |
---|---|
Sprache | englisch |
Themenwelt | Sachbuch/Ratgeber ► Gesundheit / Leben / Psychologie ► Krankheiten / Heilverfahren |
Naturwissenschaften ► Biologie | |
Technik | |
ISBN-10 | 1-4831-9151-6 / 1483191516 |
ISBN-13 | 978-1-4831-9151-5 / 9781483191515 |
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