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Phosphorylation of G Protein-Coupled Receptors: from the Barcode Hypothesis to the Flute Model

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Phosphorylation of G Protein-Coupled Receptors: from the Barcode Hypothesis to the Flute Model 1521-0111/92/3/201–210$25.00 https://doi.org/10.1124/mol.116.107839 MOLECULAR PHARMACOLOGY Mol Pharmacol 92:201–210, September 2017 Copyright ª 2017 by The Author(s) This is an open access article distributed under the CC BY-NC Attribution 4.0 International license. MINIREVIEW—MOLECULAR PHARMACOLOGY IN CHINA Phosphorylation of G Protein-Coupled Receptors: From the Barcode Hypothesis to the Flute Model Zhao Yang, Fan Yang, Daolai Zhang, Zhixin Liu, Amy Lin, Chuan Liu, Peng Xiao, Xiao Yu, and Jin-Peng Sun Downloaded from Key Laboratory Experimental Teratology of the Ministry of Education and Department of Biochemistry and Molecular Biology (Z.Y., Z.L., C.L., P.X., J.-P.S.), Department of Physiology (F.Y., X.Y.), Shandong University School of Medicine, Jinan, Shandong, People’s Republic of China; School of Pharmacy, Binzhou Medical University, Yantai, Shandong, People’s Republic of China (D.Z.); School of Medicine, Duke University, Durham, North Carolina (A.L., J.-P.S.) Received December 10, 2016; accepted February 23, 2017 molpharm.aspetjournals.org ABSTRACT Seven transmembrane G protein-coupled receptors (GPCRs) distinct functional outcomes. Our recent work using unnatural are often phosphorylated at the C terminus and on intracellular amino acid incorporation and fluorine-19 nuclear magnetic loops in response to various extracellular stimuli. Phosphoryla- resonance (19F-NMR) spectroscopy led to the flute model, tion of GPCRs by GPCR kinases and certain other kinases which provides preliminary insight into the receptor phospho- can promote the recruitment of arrestin molecules. The arrestins coding mechanism, by which receptor phosphorylation pat- critically regulate GPCR functions not only by mediating terns are recognized by an array of phosphate-binding pockets receptor desensitization and internalization, but also by redi- on arrestin and are translated into distinct conformations. rectingsignalingtoGprotein-independent pathways via These selective conformations are recognized by various at ASPET Journals on September 24, 2021 interactions with numerous downstream effector molecules. effector molecules downstream of arrestin. The phospho- Accumulating evidence over the past decade has given rise barcoding mechanism enables arrestin to recognize a wide to the phospho-barcode hypothesis, which states that ligand- range of phosphorylation patterns of GPCRs, contributing to specific phosphorylation patterns of a receptor direct its their diverse functions. Introduction activation, most GPCRs “floating” on the plasma membrane are phosphorylated at sites located on intracellular loops or Seven transmembrane-spanning G protein-coupled recep- C-terminal tails (Table 1). tors (GPCRs) comprise the largest known membrane protein Many different phosphorylation sites in different GPCRs family encoded by the human genome, and GPCRs regulate have been identified, mostly by mass spectrometry or almost all the known physiologic processes in humans by phospho-specific antibodies. By contrast, the functions of converting a broad range of extracellular stimuli (ranging receptor phosphorylation are often established by muta- from light to hormones and neurotransmitters) to intracellu- genesis both in vitro and in vivo (Budd et al., 2000; Jones lar signals (Ritter and Hall, 2009; Manglik and Kobilka, 2014; et al., 2007; Busillo et al., 2010; Bradley et al., 2016). In cells, Wisler et al., 2014; Dohlman, 2015). Upon ligand binding and the phosphorylation process is mediated by at least two classes of serine/threonine kinases, including the second This work was supported by the National Key Basic Research Program of messenger-dependent (e.g., PKA and protein kinase C China [Grant 2013CB967700]; the Fundamental Research Funds of Shandong [PKC]) and -independent kinases (i.e., GPCR kinases [GRKs]) University [Grants 2014JC029, 2016HW005]; the National Natural Science (Lefkowitz, 1998). As a classic paradigm, phosphorylation of Foundation of China [Grants 31470789, 31611540337]; the Shandong Natural Science Fund for Distinguished Young Scholars [Grants JQ201320, JQ201517]; receptors by the former type of kinases is independent of and the Program for Changjiang Scholars and Innovative Research Team in ligand binding and directly uncouples the receptors from their University [Grant IRT13028]. Z.Y. and F.Y. contributed equally to this work. cognate G proteins, leading to heterologous desensitization https://doi.org/10.1124/mol.116.107839. (Hausdorff et al., 1990). In contrast, receptor phosphorylation ABBREVIATIONS: Akt, protein kinase B; b2AR, b2-adrenergic receptor; AT1aR, angiotensin II receptor type I; BRET, bioluminescence resonance energy transfer; ERK, extracellular signal–regulated kinase; 19F-NMR, fluorine-19 nuclear magnetic resonance; GPCR, G protein-coupled receptor; GPR120, G-protein coupled receptor 120; GRK, G protein-coupled receptor kinase; GRK2, G protein-coupled receptor kinase 2, or b-adrenergic receptor kinase 1; GRK2pp, GRK2-phosphopeptides; ICL3, third intracellular loop; M3-mAChR, M3-muscarinic acetylcholine receptor; PKA, protein kinase A; PKC, protein kinase C; Thr, threonine; V2R, V2 vasopressin receptor. 201 TABLE 1. 202 Examples of receptor phosphorylation at C-terminus and corresponding functions GPCR Phosphorylation Sites Kinase Method Cell Type Function References Angiotensin II receptor T332, S335, T336, PKC/GRK Mut CHO Internalization (Thomas et al., 1998) Yang et al. type I (AT1aR) S338 S331, S338, S348 PKC Mut CHO ND (Qian et al., 1999) b2 adrenergic receptor (b2AR) S345, S346 PKA Mut Mouse L cells Desensitization (Clark et al., 1989) S355, S356, S364 GRK Mut HEK293 Desensitization (Seibold et al., 2000) S355, S356 GRK6 MS and P-SA HEK293 Desensitization and b-arrestin-mediated (Nobles et al., 2011) ERK1/2 activation In vitro b-arrestin-1 mediated SRC activation (Yang et al., 2015) T360, S364, S396, GRK2 MS and P-SA HEK293 Desensitization and internalization (Nobles et al., 2011) S401, S407, S411 In vitro b-arrestin-1 mediated clathrin recruitment (Yang et al., 2015) and internalization T384, S396, S401, GRK2 PAA In vitro ND (Fredericks et al., 1996) and S407 T384, T393, S396, GRK5 PAA In vitro ND (Fredericks et al., 1996) S401, S407, S411 C-X-C motif chemokine S324, S325 PKC/GRK6 MS and P-SA HEK293 Receptor degradation (Busillo et al., 2010) receptor type 4 (CXCR-4) S330/339, cluster GRK2/6 MS and P-SA HEK293 b-arrestin-2 recruitment (Busillo et al., 2010) S346-S352 and calcium mobilization S330/339, cluster GRK3/6 MS and P-SA HEK293 b-arrestin-1 mediated ERK1/2 activation (Busillo et al., 2010) S346-S352 Dopamine receptor D1 (DRD1) T347 ND Mut HEK293 Promote secondary phosphorylation (Kim et al., 2004) of residues within the ICL3 T360 GRK2 Mut CHO Desensitization (Lamey et al., 2002) T428, S431 GRK4 Mut HEK293T b-arrestin-1 mediated internalization (Rankin et al., 2006) S431, T439, T446 ND Mut CHO Internalization (Lamey et al., 2002) Follicle-stimulating Cluster T638-T644 GRK2 Mut HEK293 b-arrestin-mediated desensitization (Kara et al., 2006) hormone receptor (FSHR) and internalization GABAB receptor (GABABR) S783 AMPK MS and EDA Rat hippocampal Receptor recycling (Terunuma et al., 2010) neurons S867 CaMKII MS Rat hippocampal Internalization (Guetg et al., 2010) neurons S892 PKA Mut HEK293 Internalization (Couve et al., 2002) Ghrelin receptor type S349, T350 ND MS HEK293 Stabilization of the receptor-b-arrestin1/2 (Bouzo-Lorenzo et al., 2016) 1a (GHSR1a) complex S362, S363, T366 ND MS HEK293 b-arrestin1/2 binding and internalization (Bouzo-Lorenzo et al., 2016) G protein-coupled T347, S350, S357 PKC/GRK6 Mut HEK293 b-arrestin-2 recruitment and calcium (Burns et al., 2014) receptor 120 (GPR120) mobilization T347, T349, S350 ND MS CHO Flp-In cells b-arrestin-2 recruitment and Akt activation (Prihandoko et al., 2016) S357, S361 ND MS CHO Flp-In cells b-arrestin-2 recruitment and internalization (Prihandoko et al., 2016) m-opioid receptor (MOR) S356, S357, S363 PKC MS HEK293 Desensitization (Wang et al., 2002; Chen et al., 2013) T370 CaMKII MS HEK293 ND (Chen et al., 2013) S375 GRK2 MS HEK293 b-arrestin-2 recruitment and desensitization (Chen et al., 2013; Lowe et al., 2015) Thyrotropin-releasing S355, S360, S364, GRK2 Mut and P-SA CHO b-arrestin-2-mediated desensitization (Jones et al., 2007) hormone receptor (TRHR) T365 and internalization T365, T371, S383 CK2 Mut COS-1 b-arrestin-1-dependent internalization (Hanyaloglu et al., 2001) Orexin-2 receptor (OX2R) Cluster T399-S403 GRK Mut HEK293FT ND (Jaeger et al., 2014) Cluster S406-T409, GRK Mut HEK293FT Receptor-b-arrestin1/2-ubiquitin (Jaeger et al., 2014) Cluster T427-T431 complex formation AMPK, 5ʹ-AMP-activated protein kinase; CaMKII, Ca2+/calmodulin-dependent protein kinase II; CHO, Chinese hamster ovary cells; CK2, casein kinase II; COS-1, CV-1 in origin with SV40 genes monkey kidney cells; EDA, Edman degradation analysis; HEK293, human embryonic kidney 293 cells; Mut, mutagenesis; MS, mass spectrometry; ND, not determined; P-SA, phospho-specific antibody; PAA, phospho-amino acid analysis. Downloaded from from Downloaded molpharm.aspetjournals.org at ASPET Journals on September 24, 2021 24, September on Journals ASPET at Phosphorylation Barcoding of the GPCR 203 by GRKs, a kinase family consisting of seven members, is so the mechanism by which the phospho-barcode is recognized ligand-stimulation dependent and is followed by the recruit- and then converted to specific signaling remains largely ment of arrestin molecules to the receptor that sterically unknown,
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