Peptides derived from the extracellular loop of receptors: structure, mechanisms of action and application in physiology and medicine

One of the main tasks of the peptide strategy, a new direction in modern biochemistry and physiology, is the creation of selective and effective regulators of hormonal signaling systems on the basis of the peptides corresponding to functionally important regions of signal proteins. At the last years the greatest interest is connected with peptides, derivatives of the extracellular loops of receptors of the serpentine type. With these peptides the molecular basis of interaction between receptors and their ligands are studied, the new approaches for construction and testing of highly selective agonists and antagonists are developed, the etiology and pathogenesis of diseases of human and animals induced by autoimmune reactions to the extracellular loops of receptors are investigated. It is shown that peptides corresponding to the extracellular loops of the receptors and the specific antibodies to them are capable to regulate the activity of hormonal signaling systems in vitro and in vivo and can be considered as functional probes for studying of physiological functions in the norm and pathology. In the review the data obtained during the last years concerning the structures, functions, mechanisms of action and practical application ofpeptides, derivatives of the extracellular loops of serpentine type receptors, are summarized and analyzed. The prospective of their use in fundamental biology and practical medicine are discussed.     

Signaling and regulation of G protein-coupled receptors in airway smooth muscle

Signaling through G protein-coupled receptors (GPCRs) mediates numerous airway smooth muscle (ASM) functions including contraction, growth, and "synthetic" functions that orchestrate airway inflammation and promote remodeling of airway architecture. In this review we provide a comprehensive overview of the GPCRs that have been identified in ASM cells, and discuss the extent to which signaling via these GPCRs has been characterized and linked to distinct ASM functions. In addition, we examine the role of GPCR signaling and its regulation in asthma and asthma treatment, and suggest an integrative model whereby an imbalance of GPCR-derived signals in ASM cells contributes to the asthmatic state.     

Advances in the study of structure and function of G protein-coupled receptors (about awarding the Nobel Prize for Chemistry in 2012 to Robert Lefkowitz and Brian Kobilka)

The 2012 Nobel Prize for Chemistry was awarded to Robert Lefkowitz and Brian Kobilka “for study in G-protein-coupled receptors” (GPCR). In this review there are discussed and analyzed the most important discoveries of these Nobel Laureates dealing with study of structure and functions of GPCR. In the 1980s they were the first in the world to clone GPCR-the β₂-adrenergic receptor. After 20 years the group headed by B. Kobilka first obtained this receptor in the crystallic form and established its three-dimensional structure. In the course of the studies, unique approaches were developed for purifications and crystallization of other receptors. In the 1980s, R. Lefkowitz and his colleagues discovered proteins β-arrestins that regulate the signal transduction realized via GPCR. Subsequently they showed β-arrestins to be the most important participants of signal transduction and to be responsible for transduction of signal from the receptor activated with hormone to intracellular signal cascades regardless of heterotrimeric G-proteins. These and other outstanding discoveries of R. Lefkowitz and B. Kobilka have become the ground of the new field of molecular biology and pharmacology-molecular endocrinology of GPCR.     

Clathrin-dependent mechanisms of G protein-coupled receptor endocytosis

The heptahelical G protein-coupled receptor (GPCR) family includes approximately 900 members and is the largest family of signaling receptors encoded in the mammalian genome. G protein-coupled receptors elicit cellular responses to diverse extracellular stimuli at the plasma membrane and some internalized receptors continue to signal from intracellular compartments. In addition to rapid desensitization, receptor trafficking is critical for regulation of the temporal and spatial aspects of GPCR signaling. Indeed, GPCR internalization functions to control signal termination and propagation as well as receptor resensitization. Our knowledge of the mechanisms that regulate mammalian GPCR endocytosis is based predominantly on arrestin regulation of receptors through a clathrin- and dynamin-dependent pathway. However, multiple clathrin adaptors, which recognize distinct endocytic signals, are now known to function in clathrin-mediated endocytosis of diverse cargo. Given the vast number and diversity of GPCRs, the complexity of clathrin-mediated endocytosis and the discovery of multiple clathrin adaptors, a single universal mechanism controlling endocytosis of all mammalian GPCRs is unlikely. Indeed, several recent studies now suggest that endocytosis of different GPCRs is regulated by distinct mechanisms and clathrin adaptors. In this review, we discuss the diverse mechanisms that regulate clathrin-dependent GPCR endocytosis.     

A beta-arrestin binding determinant common to the second intracellular loops of rhodopsin family G protein-coupled receptors

beta-Arrestins have been shown to inhibit competitively G protein-dependent signaling and to mediate endocytosis for many of the hundreds of nonvisual rhodopsin family G protein-coupled receptors (GPCR). An open question of fundamental importance concerning the regulation of signal transduction of several hundred rhodopsin-like GPCRs is how these receptors of limited sequence homology, when considered in toto, can all recruit and activate the two highly conserved beta-arrestin proteins as part of their signaling/desensitization process. Although the serine and threonine residues that form GPCR kinase phosphorylation sites are common beta-arrestin-associated receptor determinants regulating receptor desensitization and internalization, the agonist-activated conformation of a GPCR probably reveals the most fundamental determinant mediating the GPCR and arrestin interaction. Here we identified a beta-arrestin binding determinant common to the rhodopsin family GPCRs formed from the proximal 10 residues of the second intracellular loop. We demonstrated by both gain and loss of function studies for the serotonin 2C, beta2-adrenergic, alpha2a)adrenergic, and neuropeptide Y type 2 receptors that the highly conserved amino acids, proline and alanine, naturally occurring in rhodopsin family receptors six residues distal to the highly conserved second loop DRY motif regulate beta-arrestin binding and beta-arrestin-mediated internalization. In particular, as demonstrated for the beta2 AR, this occurs independently of changes in GPCR kinase phosphorylation. These results suggest that a GPCR conformation directed by the second intracellular loop, likely using the loop itself as a binding patch, may function as a switch for transitioning beta-arrestin from its inactive form to its active receptor-binding state.     

Herpesviral G Protein-Coupled Receptors Activate NFAT to Induce Tumor Formation via Inhibiting the SERCA Calcium ATPase

G protein-coupled receptors (GPCRs) constitute the largest family of proteins that transmit signal to regulate an array of fundamental biological processes. Viruses deploy diverse tactics to hijack and harness intracellular signaling events induced by GPCR. Herpesviruses encode multiple GPCR homologues that are implicated in viral pathogenesis. Cellular GPCRs are primarily regulated by their cognate ligands, while herpesviral GPCRs constitutively activate downstream signaling cascades, including the nuclear factor of activated T cells (NFAT) pathway. However, the roles of NFAT activation and mechanism thereof in viral GPCR tumorigenesis remain unknown. Here we report that GPCRs of human Kaposi’s sarcoma-associated herpesvirus (kGPCR) and cytomegalovirus (US28) shortcut NFAT activation by inhibiting the sarcoplasmic reticulum calcium ATPase (SERCA), which is necessary for viral GPCR tumorigenesis. Biochemical approaches, entailing pharmacological inhibitors and protein purification, demonstrate that viral GPCRs target SERCA2 to increase cytosolic calcium concentration. As such, NFAT activation induced by vGPCRs was exceedingly sensitive to cyclosporine A that targets calcineurin, but resistant to inhibition upstream of ER calcium release. Gene expression profiling identified a signature of NFAT activation in endothelial cells expressing viral GPCRs. The expression of NFAT-dependent genes was up-regulated in tumors derived from tva-kGPCR mouse and human KS. Employing recombinant kGPCR-deficient KSHV, we showed that kGPCR was critical for NFAT-dependent gene expression in KSHV lytic replication. Finally, cyclosporine A treatment diminished NFAT-dependent gene expression and tumor formation induced by viral GPCRs. These findings reveal essential roles of NFAT activation in viral GPCR tumorigenesis and a mechanism of “constitutive” NFAT activation by viral GPCRs.     

Role of the SHP-2 tyrosine phosphatase in cytokine-induced signaling and cellular response

Cytokines and growth factors are important extracellular regulatory proteins. They exert their biological functions through binding to their cognate receptors on the cell surface and triggering intracellular signaling cascades. However, the intracellular signaling mechanisms of cytokines and growth factors are not well understood. Accumulating evidence has shown that protein phosphorylation and dephosphorylation carried out by protein kinases and protein phosphatases are fundamental biochemical events in intracellular signal transduction. SHP-2, a Src homology (SH) 2 domain-containing protein tyrosine phosphatase (PTP), is widely involved in a variety of signaling pathways triggered by cytokines and growth factors, including the MAP kinase, Jak-Stat, and PI3 kinase pathways. Recent studies have clearly demonstrated that this phosphatase plays an important role in transducing signals relayed from the cell surface to the nucleus, and is a critical intracellular regulator in cytokine and growth factor-induced cell survival, proliferation, and differentiation.     

GPCR signalling to the translation machinery

G protein-coupled receptors (GPCRs) are involved in most physiological processes, many of them being engaged in fully differentiated cells. These receptors couple to transducers of their own, primarily G proteins and β-arrestins, which launch intracellular signalling cascades. Some of these signalling events regulate the translational machinery to fine-tune general cell metabolism or to alter protein expression pattern. Though extensively documented for tyrosine kinase receptors, translational regulation by GPCRs is still poorly appreciated. The objective of this review paper is to address the following questions: i) is there a “GPCR signature” impacting on the translational machinery, and ultimately on the type of mRNA translated? ii) are the regulatory networks involved similar as those utilized by tyrosine kinase receptors? In particular, we will discuss the specific features of translational control mediated by GPCRs and highlight the intrinsic properties of GPCRs these mechanisms could rely on.     

Signaling through G protein coupled receptors

Heterotrimeric G proteins (Gα, Gβ/Gγ subunits) constitute one of the most important components of cell signaling cascade. G Protein Coupled Receptors (GPCRs) perceive many extracellular signals and transduce them to heterotrimeric G proteins, which further transduce these signals intracellular to appropriate downstream effectors and thereby play an important role in various signaling pathways. GPCRs exist as a superfamily of integral membrane protein receptors that contain seven transmembrane α-helical regions, which bind to a wide range of ligands. Upon activation by a ligand, the GPCR undergoes a conformational change and then activate the G proteins by promoting the exchange of GDP/GTP associated with the Gα subunit. This leads to the dissociation of Gβ/Gγ dimer from Gα. Both these moieties then become free to act upon their downstream effectors and thereby initiate unique intracellular signaling responses. After the signal propagation, the GTP of Gα-GTP is hydrolyzed to GDP and Gα becomes inactive (Gα-GDP), which leads to its re-association with the Gβ/Gγ dimer to form the inactive heterotrimeric complex. The GPCR can also transduce the signal through G protein independent pathway. GPCRs also regulate cell cycle progression. Till to date thousands of GPCRs are known from animal kingdom with little homology among them, but only single GPCR has been identified in plant system. The Arabidopsis GPCR was reported to be cell cycle regulated and also involved in ABA and in stress signaling. Here I have described a general mechanism of signal transduction through GPCR/G proteins, structure of GPCRs, family of GPCRs and plant GPCR and its role.Key words: heterotrimeric G proteins, GPCRs, seven-transmembrane receptors, signal transduction, stress signaling     

Blocking receptors on the inside: pepducin-based intervention of PAR signaling and thrombosis

Transmembrane signaling through G-protein coupled receptors (GPCRs) controls a remarkably diverse array of cellular processes including metabolism, growth, motility, adhesion, neuronal signaling, and blood coagulation. The large number of GPCRs and their important roles in normal physiology and in disease have made them the target for more than 50% of prescribed drugs. GPCR agonists and antagonists invariably act on the extracellular surface of the receptors, whereas the intracellular surface has not yet been exploited for development of new therapeutic agents. Here, we demonstrate the utility of novel cell-penetrating peptides, termed pepducins, that act as intracellular inhibitors and/or agonists of signal transference from receptor to G protein. The pepducins require the presence of their cognate receptor for activity and are highly selective for receptor type. Mutational analysis of both intact receptor and pepducins demonstrates that the cell-penetrating agonists do not activate G proteins by the same mechanism as the intact receptor i3 loop, but instead require the C-tail of the receptor. Attachment of a palmitate lipid to shorter i3 loop peptides derived from protease-activated receptors PAR1 and PAR4 created potent inhibitors of thrombin-mediated aggregation of human platelets. Infusion of the anti-PAR4 pepducin into mice extended bleeding time and protected against systemic platelet activation, consistent with the phenotype of a mouse with genetic deficiency of PAR4. These data show that pepducins may be used to ascertain the physiological roles of GPCRs and rapidly determine the potential therapeutic value of blockade of a particular signaling pathway.     

Truncated TrkB: beyond a dominant negative receptor

BDNF activates trkB receptors to regulate neuronal survival, differentiation, and proliferation. Mutations in the BDNF gene, altered BDNF expression, and altered trkB expression are associated with degenerative and psychiatric disorders. The full-length trkB receptor (trkB.tk(+)) undergoes autophosphorylation to activate intracellular signaling pathways. The truncated trkB receptor (trkB.t1) is abundantly expressed in the brain but lacks the catalytic tyrosine kinase domain. TrkB.t1 is a dominant-negative receptor that inhibits trkB.tk(+) signaling. While this is an important function of trkB.t1, it is only one of its many functions. TrkB.t1 sequesters and translocate BDNF, induces filopodia and neurite outgrowth, stimulates intracellular signaling cascades, regulates Rho GTPase signaling, and modifies cytoskeletal structures. TrkB.t1 is an active signaling molecule with regulatory effects on neurons and astrocytes.     

Nuclear localization drives α1-adrenergic receptor oligomerization and signaling in cardiac myocytes

Conventional models of G-protein coupled receptor (GPCR) signaling describe cell surface receptors binding to external ligands, such as hormones or circulating peptides, to induce intracellular signaling and a physiologic response. However, recent studies identify new paradigms indicating that GPCRs localize to and signal at the nucleus and that GPCR oligomers can influence receptor function. Previously, we reported that endogenous α1-adrenergic receptors (α1-ARs) localize to and signal at the nuclei in adult cardiac myocytes. In this study, we examined the mechanisms behind α1-AR nuclear localization and how nuclear localization impacted receptor function. We verified that endogenous α1-ARs localized to the nuclear membrane of intact nuclei isolated from wild-type adult cardiac myocytes. Next, we identified and disrupted putative nuclear localization sequences in both the α1A- and α1B-adrenergic receptors, which led to mis-localization of α1-ARs in cultured adult cardiac myocytes. Using these mutants, we demonstrated that nuclear localization was required for α1-signaling in adult cardiac myocytes. We also found that the nuclear export inhibitor leptomycin B inhibited α1-AR signaling, indicating α1-AR signaling must arise in the nucleus in adult cardiac myocytes. Finally, we found that co-localization of the α1-subtypes at the nuclei in adult cardiac myocytes facilitated the formation of receptor oligomers that could affect receptor signaling. In summary, our data indicate that α1-AR nuclear localization can drive the formation of receptor oligomers and regulate signaling in adult cardiac myocytes.     

Multiple scaffolding functions of {beta}-arrestins in the degradation of G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 (GRK2) plays a fundamental role in the regulation of G protein-coupled receptors (GPCRs), and changes in GRK2 expression levels can have an important impact on cell functions. GRK2 is known to be degraded by the proteasome pathway. We have shown previously that β-arrestins participate in enhanced kinase turnover upon GPCR stimulation by facilitating GRK2 phosphorylation by c-Src or by MAPK or by recruiting the Mdm2 E3 ubiquitin ligase to the receptor complex. In this report, we have investigated how such diverse β-arrestin scaffold functions are integrated to modulate GRK2 degradation. Interestingly, we found that in the absence of GPCR activation, β-arrestins do not perform an adaptor role for GRK2/Mdm2 association, but rather compete with GRK2 for direct Mdm2 binding to regulate basal kinase turnover. Upon agonist stimulation, β-arrestins-mediated phosphorylation of GRK2 at serine 670 by MAPK facilitates Mdm2-mediated GRK2 degradation, whereas c-Src-dependent phosphorylation would support the action of an undetermined β-arrestin-recruited ligase in the absence of GPCR activation. The ability of β-arrestins to play different scaffold functions would allow coordination of both Mdm2-dependent and -independent processes aimed at the specific modulation of GRK2 turnover in different signaling contexts.     

S-nitrosylation-regulated GPCR signaling

G protein-coupled receptors (GPCRs) are the most numerous and diverse type of cell surface receptors, accounting for about 1% of the entire human genome and relaying signals from a variety of extracellular stimuli that range from lipid and peptide growth factors to ions and sensory inputs. Activated GPCRs regulate a multitude of target cell functions, including intermediary metabolism, growth and differentiation, and migration and invasion. The GPCRs contain a characteristic 7-transmembrane domain topology and their activation promotes complex formation with a variety of intracellular partner proteins, which form basis for initiation of distinct signaling networks as well as dictate fate of the receptor itself. Both termination of active GPCR signaling and removal from the plasma membrane are controlled by protein post-translational modifications of the receptor itself and its interacting partners. Phosphorylation, acylation and ubiquitination are the most studied post-translational modifications involved in GPCR signal transduction, subcellular trafficking and overall expression. Emerging evidence demonstrates that protein S-nitrosylation, the covalent attachment of a nitric oxide moiety to specified cysteine thiol groups, of GPCRs and/or their associated effectors also participates in the fine-tuning of receptor signaling and expression. This newly appreciated mode of GPCR system modification adds another set of controls to more precisely regulate the many cellular functions elicited by this large group of receptors. This article is part of a Special Issue entitled: Regulation of cellular processes by S-nitrosylation.     

Signaling of sphingosine-1-phosphate via the S1P/EDG-family of G-protein-coupled receptors

The sphingosine-1-phosphate/Endothelial Differentiation Gene (S1P/EDG) family of G-protein-coupled receptors (GPCR) currently includes five different isoforms, which differentially regulate fundamental cellular processes such as migration, proliferation, cytoskeletal organization, adherens junction assembly and morphogenesis. Additionally, specific S1P/EDG isoforms can regulate important physiological processes such as blood vessel maturation, cardiac development and angiogenesis in vivo. Herein, we review the current state of knowledge of the expression patterns, signaling pathways and functional characteristics of the different S1P receptors. Further investigation in this field will likely improve our understanding of cardiovascular development as well as vascular diseases and may lead to novel therapeutic approaches.     

Pepducin-based intervention of thrombin-receptor signaling and systemic platelet activation

Transmembrane signaling through G protein-coupled receptors (GPCRs) controls a diverse array of cellular processes including metabolism, growth, motility, adhesion, neuronal signaling and blood coagulation. The numerous GPCRs and their key roles in both normal physiology and disease have made them the target for more than 50% of all prescribed drugs. GPCR agonists and antagonists act on the extracellular side of the receptors, whereas the intracellular surface has not yet been exploited for development of new therapeutic agents. Here, we demonstrate the utility of novel cell-penetrating peptides, termed 'pepducins', that act as intracellular inhibitors of signal transference from receptors to G proteins. Attachment of a palmitate lipid to peptides based on the third intracellular loop of protease-activated receptor 1 (PAR1) or PAR4 (refs. 3-5) yielded potent inhibitors of thrombin-mediated aggregation of human platelets. Infusion of the anti-PAR4 pepducin into mice extended bleeding time and protected against systemic platelet activation, consistent with the phenotype of PAR4-deficient mice. We show that pepducins might be used to ascertain the physiological roles of GPCRs and rapidly determine the potential therapeutic value of blockade of a particular signaling pathway.