ABSTRACT
beta-adrenergic receptors (beta-ARs), prototypic G-protein-coupled receptors (GPCRs), play a critical role in regulating numerous physiological processes. The GPCR kinases (GRKs) curtail G-protein signaling and target receptors for internalization. Nitric oxide (NO) and/or S-nitrosothiols (SNOs) can prevent the loss of beta-AR signaling in vivo, but the molecular details are unknown. Here we show in mice that SNOs increase beta-AR expression and prevent agonist-stimulated receptor downregulation; and in cells, SNOs decrease GRK2-mediated beta-AR phosphorylation and subsequent recruitment of beta-arrestin to the receptor, resulting in the attenuation of receptor desensitization and internalization. In both cells and tissues, GRK2 is S-nitrosylated by SNOs as well as by NO synthases, and GRK2 S-nitrosylation increases following stimulation of multiple GPCRs with agonists. Cys340 of GRK2 is identified as a principal locus of inhibition by S-nitrosylation. Our studies thus reveal a central molecular mechanism through which GPCR signaling is regulated.
Subject(s)
Receptors, Adrenergic, beta/metabolism , S-Nitrosothiols/metabolism , beta-Adrenergic Receptor Kinases/metabolism , Animals , Arrestins/metabolism , Cell Line , Cell Line, Tumor , Cysteine/metabolism , G-Protein-Coupled Receptor Kinase 2 , Homeostasis , Humans , Lung/metabolism , Mice , Myocardium/metabolism , Nitric Acid/metabolism , Phosphorylation , Signal Transduction , beta-Adrenergic Receptor Kinases/chemistry , beta-ArrestinsABSTRACT
MOTIVATION: Epithelial Na(+) channels (ENaC) mediate the transport of sodium (Na) across epithelia in the kidney, gut and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4-2. These ligases bind to proline-rich motifs (PY motifs) present in the C-termini of ENaC subunits. Loss of this inhibition leads to hypertension. We have previously reported that ENaC channels are maintained in the active state by the G protein coupled receptor kinase, GRK2. The enzyme has been implicated in the development of essential hypertension [R. D. Feldman (2002) Mol. Pharmacol., 61, 707-709]. Additional findings in our lab pointed towards a possible role for GRK2 in the phosphorylation and inactivation of Nedd4-2. RESULTS: We have predicted GRK2 phosphorylation sites on Nedd4-2 by combining sequence analysis, homology modeling and surface accessibility calculations. A total of 24 potential phosphorylation sites were predicted by sequence analysis. Of these, 16 could be modeled using homology modeling and 6 of these were found to have sufficient surface exposure to be accessible to the GRK2 enzyme responsible for the phosphorylation of Nedd4-2. The method provides an ordered list of the most probable GRK2 phosphorylation sites on Nedd4-2 providing invaluable guidance to future experimental studies aimed at mutating certain Nedd4-2 residues in order to prevent phosphorylation by GRK2. The method developed could be applied in a wide variety of biological applications involving the binding of one molecule to a protein. The relative effectiveness of the technique is determined mainly by the quality of the homology model built for the protein of interest. CONTACT: jarthur@med.usyd.edu.au
Subject(s)
Algorithms , Models, Chemical , Models, Molecular , Sequence Analysis, Protein/methods , Ubiquitin-Protein Ligases/chemistry , beta-Adrenergic Receptor Kinases/chemistry , Amino Acid Sequence , Artificial Intelligence , Binding Sites , Computer Simulation , Endosomal Sorting Complexes Required for Transport , G-Protein-Coupled Receptor Kinase 2 , Molecular Sequence Data , Nedd4 Ubiquitin Protein Ligases , Phosphorylation , Protein Binding , Protein Interaction Mapping/methods , Sequence Alignment/methods , Sequence Homology, Amino AcidABSTRACT
Hypothalamic CRF stimulates synthesis and secretion of ACTH via CRF receptor type 1 (CRFR1) in the anterior pituitary gland. After agonist-activated stimulation of receptor signaling, CRFR1 is down-regulated and desensitized. Generally, it is thought that G protein-coupled receptors may be desensitized by G protein-coupled receptor kinases (GRKs). However, the role of GRKs in corticotropic cells has not been determined. In this study we focused on involvement of GRKs in desensitization of CRFR1 by CRF in corticotropic cells. We found that GRK2 (but not GRK3) mRNA and protein were expressed in rat anterior pituitary cells and AtT-20 cells (a line of mouse corticotroph tumor cells). To determine the role of GRK2 in CRF-induced desensitization of CRFR1 in mouse corticotrophs, AtT-20 cells were transfected with a dominant-negative mutant GRK2 construct. CRF desensitized the cAMP-dependent response by CRFR1. Desensitization of CRFR1 by CRF was significantly less in AtT-20 cells transfected with the dominant-negative mutant GRK2 construct compared with desensitization in control (an empty vector-transfected) AtT-20 cells. Furthermore, pretreatment with a protein kinase A inhibitor also partially blocked desensitization of CRFR1 by CRF. These results suggest that GRK2 is involved in CRF-induced desensitization of CRFR1 in AtT-20 cells, and the protein kinase A pathway may also have an important role in desensitization of CRFR1 by CRF seen in corticotropic cells.
Subject(s)
Corticotropin-Releasing Hormone/pharmacology , Hypothalamus/physiology , Receptors, Corticotropin-Releasing Hormone/physiology , beta-Adrenergic Receptor Kinases/metabolism , Adrenocorticotropic Hormone/metabolism , Animals , Cyclic AMP/physiology , DNA Primers , G-Protein-Coupled Receptor Kinase 2 , G-Protein-Coupled Receptor Kinase 3 , G-Protein-Coupled Receptor Kinase 4 , Male , Protein Serine-Threonine Kinases/metabolism , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , beta-Adrenergic Receptor Kinases/chemistryABSTRACT
G protein-coupled receptor kinase 2 (GRK2) plays a key role in the desensitization of G protein-coupled receptor signaling by phosphorylating activated heptahelical receptors and by sequestering heterotrimeric G proteins. We report the atomic structure of GRK2 in complex with Galphaq and Gbetagamma, in which the activated Galpha subunit of Gq is fully dissociated from Gbetagamma and dramatically reoriented from its position in the inactive Galphabetagamma heterotrimer. Galphaq forms an effector-like interaction with the GRK2 regulator of G protein signaling (RGS) homology domain that is distinct from and does not overlap with that used to bind RGS proteins such as RGS4.
