Selective inactivation of guanine-nucleotide-binding regulatory protein (G-protein) α and βγ subunits by urea

2001 ◽  
Vol 354 (2) ◽  
pp. 337-344 ◽  
Author(s):  
William K. LIM ◽  
Richard R. NEUBIG
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Regulatory Protein ◽  
Significant Loss ◽  
Membrane Extraction ◽  
Α Subunit ◽  
Mammalian Cell Lines ◽  
Membrane Bound ◽  
Guanine Nucleotide Binding ◽  
G Protein Coupled

G-protein-coupled receptors activate signal-transducing G-proteins, which consist of an α subunit and a βγ dimer. Membrane extraction with 5–7M urea has been used to uncouple receptors from endogenous G-proteins to permit reconstitution with purified G-proteins. We show that αi subunits are inactivated with 5M urea whereas the βγ dimer requires at least 7M urea for its inactivation. There is no significant loss of receptors. Surprisingly, Western-blot analysis indicates that the urea-denatured αi subunit remains mostly membrane-bound and that β is only partially removed. After 7M urea treatment, both αi1 and βγ subunits are required to restore high-affinity agonist binding and receptor-catalysed guanosine 5′-[γ-thio]triphosphate binding. We demonstrate the generality of this approach for four Gi-coupled receptors (α2A-adrenergic, adenosine A1, 5-hydroxytryptamine1A and µ-opioid) expressed in insect cells and two mammalian cell lines. Thus a selectivity of urea for G-protein α versus βγ subunits is established in both concentration and mechanism.

New thoughts on the role of the βγ subunit in G protein signal transduction

2000 ◽  
Vol 78 (5) ◽  
pp. 537-550 ◽  
Author(s):  
Barbara Vanderbeld ◽  
Gregory M Kelly
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
G Proteins ◽  
G Protein Coupled Receptors ◽  
Limited Information ◽  
Α Subunit ◽  
Downstream Effectors ◽  
Receptor Signal ◽  
G Protein Coupled

Heterotrimeric G proteins are involved in numerous biological processes, where they mediate signal transduction from agonist-bound G-protein-coupled receptors to a variety of intracellular effector molecules and ion channels. G proteins consist of two signaling moieties: a GTP-bound α subunit and a βγ heterodimer. The βγ dimer, recently credited as a significant modulator of G-protein-mediated cellular responses, is postulated to be a major determinant of signaling fidelity between G-protein-coupled receptors and downstream effectors. In this review we have focused on the role of βγ signaling and have included examples to demonstrate the heterogeneity in the heterodimer composition and its implications in signaling fidelity. We also present an overview of some of the effectors regulated by βγ and draw attention to the fact that, although G proteins and their associated receptors play an instrumental role in development, there is rather limited information on βγ signaling in embryogenesis.Key words: G protein, βγ subunit, G-protein-coupled receptor, signal transduction, adenylyl cyclase.

Is Receptor Cross-Regulation in Human Heart Caused by Alterations in Cardiac Guanine Nucleotide-Binding Proteins?

1993 ◽  
Vol 85 (4) ◽  
pp. 393-399 ◽  
Author(s):  
A. Ferro ◽  
C. Plumpton ◽  
M. J. Brown
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Atrial Appendage ◽  
Right Atrial ◽  
Α Subunit ◽  
Guanine Nucleotide Binding ◽  
Quantitative Changes ◽  
Right Atrial Appendage ◽  
Nucleotide Binding Proteins ◽  
Guanine Nucleotide Binding Proteins

1. Guanine nucleotide-binding proteins (G-proteins) play a central role in signal transduction between a wide variety of cell-surface receptors and intracellular second messenger systems. Recently, we and others have demonstrated that cross-regulation can occur between a variety of G-protein-linked receptors in human heart. Chronic β1-adrenoceptor blockade gives rise to sensitization of β2-adrenoceptor and of 5HT4-receptor responses, both of which are mediated via stimulation of adenylate cyclase through stimulatory G-proteins (Gs), and also gives rise to desensit-ization of muscarinic M2-receptor responses, which inhibit adenylate cyclase through inhibitory G-proteins (Gi). 2. In order to investigate whether these effects are due to quantitative changes in cardiac G-protein isoforms, we measured their abundance in right atrial appendage from patients taking or not taking β1-adrenoceptor antagonists, by immunoblotting. 3. Samples of right atrial appendage homogenate were subjected to SDS/PAGE, and proteins were electroblotted on to nitrocellulose membranes. These were then probed with specific anti-G protein anti-sera, and binding was revealed by means of a secondary antibody labelled with alkaline phosphatase and using a chromogenic substrate. The resulting bands were quantified by laser densitometry. 4. No quantitative differences were detected, between these two groups of patients, in the amounts of α-subunit of ‘long’ or ‘short’ Gs isoforms (GsαL and GsαS), or in the amounts of Gi 1 + 2 α-subunit (Giα1 + 2). Nor was any difference found in the abundance of the β-subunit of G-proteins. No ‘other’ G-protein (Go) was detectable in these samples by immunoblotting. 5. We conclude that the phenomenon of receptor cross-regulation which we have previously observed in human right atrial appendage is unlikely to be explained by quantitative changes at the G-protein level.

scholarly journals Role of G-proteins in the differentiation of epiphyseal chondrocytes

2014 ◽  
Vol 53 (2) ◽  
pp. R39-R45 ◽  
Author(s):  
Andrei S Chagin ◽  
Henry M Kronenberg
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Growth Plate ◽  
Current Knowledge ◽  
Postnatal Growth ◽  
Chondrocyte Differentiation ◽  
Growth Plate Chondrocytes ◽  
Α Subunit ◽  
G Protein Coupled

Herein, we review the regulation of differentiation of the growth plate chondrocytes by G-proteins. In connection with this, we summarize the current knowledge regarding each family of G-protein α subunit, specifically, Gαs, Gαq/11, Gα12/13, and Gαi/o. We discuss different mechanisms involved in chondrocyte differentiation downstream of G-proteins and different G-protein-coupled receptors (GPCRs) activating G-proteins in the epiphyseal chondrocytes. We conclude that among all G-proteins and GPCRs expressed by chondrocytes, Gαshas the most important role and prevents premature chondrocyte differentiation. Receptor for parathyroid hormone (PTHR1) appears to be the major activator of Gαsin chondrocytes and ablation of either one leads to accelerated chondrocyte differentiation, premature fusion of the postnatal growth plate, and ultimately short stature.

Visualizing Signal Transduction: Receptors, G-Proteins, and Adenylate Cyclases

1996 ◽  
Vol 91 (5) ◽  
pp. 527-537 ◽  
Author(s):  
Carmen W. Dessauer ◽  
Bruce A. Posner ◽  
Alfred G. Gilman
Keyword(s):  
Signal Transduction ◽  
Adenylate Cyclase ◽  
G Protein ◽  
G Proteins ◽  
Crystallographic Analysis ◽  
Α Subunit ◽  
Signalling Network ◽  
Great Insight ◽  
Guanine Nucleotide Binding ◽  
Retinal Cgmp Phosphodiesterase

1. The first glimpses of heterotrimeric G-proteins came with the discoveries of the ubiquitous adenylate cyclase activator, Gs, and the specialized retinal cGMP phosphodiesterase activator, Gt or transducin. The model that evolved for regulation of adenylate cyclase activity by G-proteins soon proved to be a general paradigm for a large number of signalling pathways. Although many different G-proteins interact with a diverse array of receptors and effectors, each is composed of a guanine-nucleotide-binding α-subunit and a tightly associated complex of a β- and a γ-subunit. 2. Receptors catalyse the activation of G-proteins by promoting exchange of GDP for GTP, while G-proteins catalyse their own deactivation as a result of their intrinsic GTPase activity. Crystallographic analysis has described several of the various conformational states that G-proteins undergo as they are activated and deactivated and has provided great insight into the kinetic models of G-protein-mediated signal transduction. 3. The regulation of adenylate cyclase has proven to be intriguing and complex. Gsα activates all forms of mammalian adenylate cyclase; other G-proteins (Gi, Go and Gz) inhibit certain isoforms of the enzyme. The discovery of new isoforms of adenylate cyclase has revealed synergistic and conditional mechanisms of regulation. These include activation or inhibition by the G-protein βγ-subunit complex, activation by Ca2+-calmodulin, and phosphorylation by protein kinases. The large number of receptors, G-proteins and adenylate cyclases provides a complex signalling network that integrates and interprets a multitude of convergent inputs.

scholarly journals The puzzling uniqueness of the heterotrimeric G15 protein and its potential beyond hematopoiesis

2010 ◽  
Vol 44 (5) ◽  
pp. 259-269 ◽  
Author(s):  
Flavia Giannone ◽  
Giorgio Malpeli ◽  
Veronica Lisi ◽  
Silvia Grasso ◽  
Priyanka Shukla ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Physiological Role ◽  
Cell Types ◽  
Biochemical Properties ◽  
Membrane Receptors ◽  
General Mechanism ◽  
Specific Cell ◽  
Α Subunit ◽  
G Protein Coupled

Heterotrimeric G proteins transduce the signals of the largest family of membrane receptors (G protein-coupled receptors, GPCRs) hence triggering the activation of a wide variety of physiological responses. G15 is a G protein characterized by a number of functional peculiarities that make its signaling exceptional: 1) it can couple a variety of Gs-, Gi/o-, and Gq-linked receptors to phospholipase C activation; 2) relatively to other G proteins, it is poorly affected by β-arrestin-dependent desensitization, the general mechanism that regulates GPCR function and 3) at the protein level, its expression is only detected in highly specific cell types (hematopoietic and epithelial cells). G15 α-subunit displays unique structural and biochemical properties, and is phylogenetically the most recent and divergent component of the Gαq/11 subfamily. All these aspects shed a mysterious light on G15 biological role, which remains substantially elusive. Thus, far, G15 signaling has been analyzed in the context of hematopoiesis. Here, we highlight observations supporting the view that G15 functions may extend further beyond the immune system. In addition, we describe puzzling aspects of G15 signaling that offer a novel perspective in the understanding of its physiological role.

Hereditary and acquired defects in signaling through the hormone-receptor-G protein complex

1994 ◽  
Vol 266 (2) ◽  
pp. F163-F174
Author(s):  
J. R. Raymond
Keyword(s):  
G Protein ◽  
Neutralizing Antibodies ◽  
Second Messengers ◽  
Regulatory Protein ◽  
Extracellular Signals ◽  
Extracellular Signaling ◽  
Genes Encoding ◽  
Membrane Bound ◽  
Guanine Nucleotide Binding ◽  
Signaling Components

Extracellular signals reach the interior of cells as second messengers through intermediary transducers located within or closely associated with the plasma membrane. One of the most common pathways involves the interaction of the extracellular signaling element with a membrane-bound receptor and guanine nucleotide-binding regulatory protein (G protein). Inherited and acquired defects that alter the function of the first messenger (hormone, neurotransmitter, autacoid, or paracrine substance) or either of the transducing components (G protein-coupled receptor or G protein) can lead to defective signaling and, ultimately, disease. Clinically relevant examples of defects of all three of those signaling components have recently been described. These can take the form of inherited or sporadic mutations of the genes encoding the various signaling components, or of neutralizing antibodies against those components. The purpose of this review is to summarize how acquired or inherited defects in each of those pathways might lead to diseases.

scholarly journals Rapid Establishment of G-Protein-Coupled Receptor–Expressing Cell Lines by Site-Specific Integration

2011 ◽  
Vol 16 (3) ◽  
pp. 323-331 ◽  
Author(s):  
Roland Schucht ◽  
Simon Lydford ◽  
Lisa Andzinski ◽  
Jeannette Zauers ◽  
James Cooper ◽  
...  
Keyword(s):  
Cell Line ◽  
G Protein ◽  
Cell Lines ◽  
Reporter Protein ◽  
Mammalian Cell Lines ◽  
Site Specific Integration ◽  
Efficient Integration ◽  
Membrane Bound ◽  
G Protein Coupled ◽  
Dna Recombinase

The establishment of mammalian cell lines reliably expressing G-protein-coupled receptors (GPCRs) can be a tedious and often time-consuming process. A strategy has been developed to allow the rapid production of such cell lines. The first step of this approach was the generation of a specialized master cell line, characterized by optimized stable expression of a membrane-bound reporter protein. In the second step, this reporter gene was exchanged for that of the GPCR of interest by a DNA recombinase “cut-and-paste” engineering step. It has been demonstrated that the resulting GPCR cell lines inherit the advantages of the master cell line, expressing the GPCR in a homogeneous and stable manner. The case studies presented demonstrate the functionality of the established GPCR cell lines, and most important, because of the highly efficient integration event, these recombinant GPCR-expressing cell lines were generated within a timeframe of 2 to 4 weeks. The advantages of this cut-and-paste approach versus other strategies such as Flp-In or Jump-In are compared.

Regulation of heartbeat by G protein-coupled ion channels

1990 ◽  
Vol 259 (6) ◽  
pp. H1621-H1628 ◽  
Author(s):  
A. M. Brown
Keyword(s):  
Ion Channels ◽  
G Protein ◽  
G Proteins ◽  
Surprising Result ◽  
Muscarinic Response ◽  
Cellular Factors ◽  
G Protein Alpha Subunit ◽  
Guanine Nucleotide Binding ◽  
Second Messenger Pathways ◽  
G Protein Coupled

The coupling of ion channels to receptors by G proteins is the subject of this American Physiological Society Walter B. Cannon Memorial "Physiology in Perspective" Lecture. This subject is particularly appropriate because it includes a molecular explanation of a homeostatic mechanism involving the autonomic nervous system and the latter subject preoccupied Dr. Cannon during most of his career. With the use of reconstitution methods, we and others have shown that heterotrimeric guanine nucleotide-binding (G) proteins couple receptors to ion channels by both membrane-delimited, direct pathways and cytoplasmic second messenger pathways. Furthermore, one set of receptors may be coupled to as many as three different sets of ion channels to form networks. Dual G protein pathways lead to the prediction of biphasic ion current responses in cell signaling, and this prediction was confirmed. In sinoatrial pacemaker cells, the pacemaking hyperpolarization-activated inward current (If) is directly regulated by the G proteins Gs and Go, and the two can act simultaneously. This could explain the classical observation that vagal inhibition of heart rate is greater during sympathetic stimulation. Because deactivation of the muscarinic response occurs much faster than the G protein alpha-subunit hydrolyzes guanosine 5'-triphosphate, we looked for accessory cellular factors. A surprising result was that the small monomeric ras G protein blocked the muscarinic pathway. The significance of this observation is unknown, but it appears that small and large G proteins may interact in ion channel signaling pathways.

scholarly journals Every Detail Matters. That Is, How the Interaction between Gα Proteins and Membrane Affects Their Function

Membranes ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 222
Author(s):  
Agnieszka Polit ◽  
Paweł Mystek ◽  
Ewa Błasiak
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
G Proteins ◽  
Lipid Membranes ◽  
Signaling Proteins ◽  
Heterotrimeric G Proteins ◽  
Membrane Attachment ◽  
The Individual ◽  
Multicellular Organisms ◽  
G Protein Coupled

In highly organized multicellular organisms such as humans, the functions of an individual cell are dependent on signal transduction through G protein-coupled receptors (GPCRs) and subsequently heterotrimeric G proteins. As most of the elements belonging to the signal transduction system are bound to lipid membranes, researchers are showing increasing interest in studying the accompanying protein–lipid interactions, which have been demonstrated to not only provide the environment but also regulate proper and efficient signal transduction. The mode of interaction between the cell membrane and G proteins is well known. Despite this, the recognition mechanisms at the molecular level and how the individual G protein-membrane attachment signals are interrelated in the process of the complex control of membrane targeting of G proteins remain unelucidated. This review focuses on the mechanisms by which mammalian Gα subunits of G proteins interact with lipids and the factors responsible for the specificity of membrane association. We summarize recent data on how these signaling proteins are precisely targeted to a specific site in the membrane region by introducing well-defined modifications as well as through the presence of polybasic regions within these proteins and interactions with other components of the heterocomplex.

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