PKA R subunits mediate synergy amone GPCR cascades (original) (raw)
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The Structural Basis of G-Protein-Coupled Receptor Signaling (Nobel Lecture)
Angewandte Chemie International Edition, 2013
Complex organisms require a sophisticated communication network to maintain homeostasis. Cells from different parts of our bodies communicate with each other using chemical messengers in the form of hormones and neurotransmitters. Cells process information encoded in these chemical messages using G protein coupled receptors (GPCRs) located in the plasma membrane. GPCRs are also mediate communication with the outside world. The senses of sight, smell and taste are mediated by GPCRs. G protein coupled receptors (GP-CRs) are nature's most versatile chemical sensors. There are over 800 GPCRs in the human genome and they respond to a broad spectrum of chemical entities ranging from photons, protons and calcium ions, and small organic molecules (including odorants and neurotransmitters), to peptides and glycoproteins. The classical role of a GPCR is to detect the presence of an extracellular agonist, transmit the information across the plasma membrane, and activate a cytoplasmic heterotrimeric G protein, leading to modulation of downstream effector proteins. Taking the human β 2 AR as an example, binding of adrenaline leads to activation of Gαs, stimulation of adenylyl cyclase, cAMP accumulation, PKA activation, and phosphorylation of proteins involved in cell metabolism (Fig. 1). However, a wealth of research has now demonstrated that many GPCRs have more complex signaling repertoires. For example, the β 2 AR couples to both Gαs and Gαi in cardiac myocytes (Xiao et al. 1999), and can also signal through MAP kinase pathways in a G protein-independent manner via arrestin (Azzi et al. 2003;
Molecular and Cellular Biology, 2000
The two yeast pheromone receptors, the a and α-factor receptors, share many functional similarities: both G protein-coupled receptors couple to the same downstream signal transduction pathway, and both receptors undergo feedback regulation involving increased phosphorylation on their C-terminal domains in response to ligand challenge. The present work, which focuses on the signaling mechanism controlling this feedback phosphorylation, indicates one striking difference. While the α-factor-induced phosphorylation of the α-factor receptor does not require activation of the downstream G protein-directed signaling pathway (B. Zanolari, S. Raths, B. Singer-Kruger, and H. Riezman, Cell 71:755–763, 1992), the a-factor-induced phosphorylation of the a-factor receptor (Ste3p) clearly does. Induced Ste3p phosphorylation was blocked in cells with disruptions of various components of the pheromone response pathway, indicating a requirement of pathway components extending from the G protein down ...
Journal of Biological Chemistry, 2005
The A 2A adenosine receptor is a prototypical G s-coupled receptor, but it also signals, e.g. to mitogen-activated protein (MAP) kinase, via a pathway that is independent of heterotrimeric G proteins. Truncation of the carboxyl terminus affects the strength of the signal through these alternative pathways. In a yeast two-hybrid interaction hunt, we screened a human brain library for proteins that bound to the juxtamembrane portion of the carboxyl terminus of the A 2A receptor. This approach identified ARNO/cytohesin-2, a nucleotide exchange factor for the small (monomeric) G proteins of the Arf (ADP-ribosylation factor) family, as a potential interaction partner. We confirmed a direct interaction by mutual pull down (of fusion proteins expressed in bacteria) and by immunoprecipitation of the proteins expressed in mammalian cells. To circumvent the long term toxicity associated with overexpression of ARNO/cytohesin-2, we created stable cell lines that stably expressed the A 2A receptor and where ARNO/cytohesin-2 or the dominant negative version E156K-ARNO/ cytohesin-2 was inducible by mifepristone. Cyclic AMP accumulation induced by an A 2A-specific agonist was neither altered by ARNO/cytohesin-2 nor by the dominant negative version. This was also true for agonistinduced desensitization. In contrast, expression of dominant negative E156K-ARNO/cytohesin-2 and of dominant negative T27N-Arf6 abrogated the sustained phase of MAP kinase stimulation induced by the A 2A receptor. We therefore conclude that ARNO/cytohesin-2 is required to support the alternative, heterotrimeric G protein-independent, signaling pathway of A 2A receptor, which is stimulation of MAP kinase.
Journal of Biological Chemistry, 1998
The classical paradigm for G protein-coupled receptor (GPCR) signal transduction involves the agonistdependent interaction of GPCRs with heterotrimeric G proteins at the plasma membrane and the subsequent generation, by membrane-localized effectors, of soluble second messengers or ion currents. Termination of GPCR signals follows G protein-coupled receptor kinase (GRK)-and -arrestin-mediated receptor uncoupling and internalization. Here we show that these paradigms are inadequate to account for GPCR-mediated, Ras-dependent activation of the mitogen-activated protein (MAP) kinases Erk1 and -2. In HEK293 cells expressing dominant suppressor mutants of -arrestin or dynamin,  2 -adrenergic receptor-mediated activation of MAP kinase is inhibited. The inhibitors of receptor internalization specifically blocked Raf-mediated activation of MEK. Plasma membrane-delimited steps in the GPCRmediated activation of the MAP kinase pathway, such as tyrosine phosphorylation of Shc and Raf kinase activation by Ras, are unaffected by inhibitors of receptor internalization. Thus, GRKs and -arrestins, which uncouple GPCRs and target them for internalization, function as essential elements in the GPCR-mediated MAP kinase signaling cascade.
Molecular microbiology, 2003
G protein-coupled receptors (GPCRs) help to regulate the physiology of all the major organ systems. They respond to a multitude of ligands and activate a range of effector proteins to bring about the appropriate cellular response. The choice of effector is largely determined by the interaction of individual GPCRs with different G proteins. Several factors influence this interaction, and a better understanding of the process may enable a more rational approach to identifying compounds that affect particular signalling pathways. A number of systems have been developed for the analysis of GPCRs. All provide useful information, but the genetic amenability and relative simplicity of yeast makes them a particularly attractive option for ligand identification and pharmaceutical screening. Many, but not all, GPCRs are functional in the budding yeast Saccharomyces cerevisiae , and we have developed reporter strains of the fission yeast Schizosaccharomyces pombe as an alternative host. To provide a more generic system for investigating GPCRs, we created a series of yeast-human G a a a atransplants, in which the last five residues at the Cterminus of the yeast G a a a a -subunit are replaced with the corresponding residues from different human G proteins. These enable GPCRs to be coupled to the Sz. pombe signalling machinery so that stimulation with an appropriate ligand induces the expression of a signal-dependent lacZ reporter gene. We demonstrate the specificity of the system using corticotropin releasing factor (CRF) and CRF-related peptides on two CRF receptors. We find that different combina-tions of ligand and receptor activate different G a a a atransplants, and the specificity of the coupling is similar to that in mammalian systems. Thus, CRF signalled through the Gs-and Gi-transplants, consistent with its regulation of adenylate cyclase, and was more active against the CRF-R 1A receptor than against the CRF-R 2B receptor. In contrast, urocortin II and urocortin III were selective for the CRF-R 2B receptors. Furthermore, urocortin, but not CRF, induced signalling through the CRF-R 1A receptor and the Gq-transplant. This is the first time that human GPCRs have been coupled to the signalling pathway in Sz. pombe , and the strains described in this study will complement the other systems available for studying this important family of receptors.
Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling
Proceedings of the National Academy of Sciences of the United States of America, 2016
Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an ...
G protein-coupled receptor adaptation mechanisms
Seminars in Cell & Developmental Biology, 1998
G protein-coupled receptors GPCRs transduce extracellular signals that modulate the activity of a wide variety of biological processes, such as neurotransmission, chemoattraction, cardiac function, olfaction, and vision. However, GPCR signalling desensitizes rapidly as the consequence of receptor phosphorylation. G protein-coupled receptor kinasemediated receptor phosphorylation promotes the binding of -arrestin proteins, which not only uncouple GPCRs from their cognate heterotrimeric G protein, but also target them ( ) for endocytosis. The sequestration endocytosis of desensitized GPCRs to endosomes is required for their dephosphorylation and subsequent resensitization to their pre-ligand exposed state. This review concentrates on the mechanisms underlying GPCR desensitization and resensitization.
Microbial Cell, 2021
A major signal transduction pathway regulating cell growth and many associated physiological properties as a function of nutrient availability in the yeast Saccharomyces cerevisiae is the protein kinase A (PKA) pathway. Glucose activation of PKA is mediated by G-protein coupled receptor (GPCR) Gpr1, and secondary messenger cAMP. Other nutrients, including nitrogen, phosphate and sulfate, activate PKA in accordingly-starved cells through nutrient transceptors, but apparently without cAMP signaling. We have now used an optimized EPAC-based fluorescence resonance energy transfer (FRET) sensor to precisely monitor in vivo cAMP levels after nutrient addition. We show that GPCR-mediated glucose activation of PKA is correlated with a rapid transient increase in the cAMP level in vivo, whereas nutrient transceptor-mediated activation by nitrogen, phosphate or sulfate, is not associated with any significant increase in cAMP in vivo. We also demonstrate direct physical interaction between the...
Journal of Biological Chemistry, 2001
The 2 adrenergic receptor (2AR) undergoes desensitization by a process involving its phosphorylation by both protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs). The protein kinase A-anchoring protein AKAP79 influences 2AR phosphorylation by complexing PKA with the receptor at the membrane. Here we show that AKAP79 also regulates the ability of GRK2 to phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced phosphorylation of both the 2AR and a mutant of the receptor that cannot be phosphorylated by PKA (2AR/PKA؊). Mutants of AKAP79 that do not bind PKA or target to the 2AR markedly inhibit phosphorylation of 2AR/PKA؊. We show that PKA directly phosphorylates GRK2 on serine 685. This modification increases G␥ subunit binding to GRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor. Abrogation of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or by expression of a dominant negative AKAP79 mutant reduces GRK2-mediated translocation to 2AR and phosphorylation of agonist-occupied 2AR, thus reducing subsequent receptor internalization. Agonist-stimulated PKA-mediated phosphorylation of GRK2 may represent a mechanism for enhancing receptor phosphorylation and desensitization.