Biased S1PR1 Signaling in B Cells Subverts Responses to Homeostatic Chemokines, Severely Disorganizing Lymphoid Organ Architecture This information is current as of October 3, 2021. Il-Young Hwang, Chung Park, Kathleen Harrison and John H. Kehrl J Immunol published online 23 September 2019 http://www.jimmunol.org/content/early/2019/09/20/jimmun ol.1900678 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2019/09/20/jimmunol.190067 Material 8.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 3, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 23, 2019, doi:10.4049/jimmunol.1900678 The Journal of Immunology Biased S1PR1 Signaling in B Cells Subverts Responses to Homeostatic Chemokines, Severely Disorganizing Lymphoid Organ Architecture Il-Young Hwang, Chung Park, Kathleen Harrison, and John H. Kehrl Ligand-engaged chemoattractant receptors trigger Gai subunit nucleotide exchange, stimulating the activation of downstream effector molecules. Activated chemoattractant receptors also dock G protein–coupled receptor kinases (GRKs) that help mediate receptor desensitization. In this study, we show that the B cell–specific loss of GRK2 severely disrupts B cell trafficking and immune cell homeostasis. The GRK2 deficiency in developing murine B cells leads to a severe immune phenotype, including a major reduction of bone marrow IgD+ cells, splenomegaly with a loss of white pulp and grossly expanded red pulp, a deficit of Peyer patches, and small lymph nodes with marked reductions in B cell numbers. The major phenotypes in these mice arise from excessive S1PR1 signaling combined with inadequate homeostatic chemokine receptor signaling. CXCL13 signaling is the most Downloaded from severely compromised. In B cells, our data also indicate that S1PR1 signals constitutively, as blocking S1PR1 signaling with an S1PR1 antagonist enhanced CXCL13-triggered wild-type B cell migration. Furthermore, blocking S1PR1 signaling in the GRK2-deficient B cells partially corrected their poor response to chemokines. Treating mice lacking GRK2 expression in their B cells with an S1PR1 antagonist partially normalized B cell trafficking into lymph node and splenic follicles. These findings reveal the critical interdependence of Gai-linked signaling pathways in controlling B lymphocyte trafficking. The Journal of Immunology, 2019, 203: 000–000. http://www.jimmunol.org/ n array of chemoattractant receptors modulates the Activated G protein–coupled receptors (GPCRs) also recruit positioning and trafficking of B lymphocytes (1–3). GPCR kinases (GRKs). They are proposed to dock the 20-aa A Each of these receptors use Gai proteins to activate N-terminal a helix of GRKs much like they bind the C-terminal downstream effector molecules (4, 5). Mice whose lymphocytes a5 helix of the Ga subunit of GDP-bound heterotrimeric G pro- lack Gai2 and Gai3 proteins or that possess Gai proteins un- teins (8). Consequently, GRKs and heterotrimeric G proteins able to interact with regulator of G protein–signaling proteins compete for the same site on the activated receptor. Typically, G exhibit severe defects in chemoattractant receptor signaling protein binding precludes GRK binding and vice versa. However, by guest on October 3, 2021 and in the organization of B cell compartments (5, 6). By specific receptor conformations may favor the binding of one adopting an active conformation ligand, engaged receptors protein versus the other. Structural studies indicate that activated recruit heterotrimeric G proteins (7). The activated receptor GPCRs adopt a dynamic conformational landscape rather a sin- opens a cleft between the helical and ras domains in the Gai gle “active” conformation (7). Based on the structural analysis subunit, which facilitates GDP release, allowing GTP to bind. of GRK5/b2–adrenergic receptors interactions, receptor docking The GTP-bound Gai and -freed Gbg subunits can then stimulate causes a GRK conformation change reorienting the catalytic do- the activity of downstream effector molecules. Precise regulation main increasing catalytic activity (9). This contrasts with most of Gbg– and GTP–Gai signaling coordinate an assortment of other protein kinases, whose catalytic activity depends upon a post- signaling pathways that enable B cell integrin activation and di- translational modification such as phosphorylation. Once recruited rected migration. and activated, GRKs phosphorylate GPCRs, typically on their C-terminal serine/threonine residues. An activated, phosphory- lated receptor becomes a target for b-arrestins, whose recruitment B Cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, leads to receptor desensitization and receptor endocytosis. Fol- MD 20892 lowing endocytosis, the receptor is degraded or recycles to the ORCIDs: 0000-0002-7819-5333 (C.P.); 0000-0002-6526-159X (J.H.K.). plasma membrane (10). Received for publication June 18, 2019. Accepted for publication August 29, 2019. The GRKs are the serine/threonine protein kinases most related This work was supported by an intramural program of the National Institute of to the AGC kinase subfamily (8). They have a central catalytic Allergy and Infectious Disease. domain located within a regulator of G protein–signaling homology I.-Y.H. designed and performed the experiments, helped analyze the data; C.P. did the (RH) domain, which is flanked by a short N-terminal a-helical intravital imaging and confocal microscopy; K.H. managed the mice and helped with domain and a variable C-terminal lipid-binding region. Among the the experiments; J.H.K. conceived the experiments, helped in data analysis, and wrote the manuscript. seven mammalian GRKs, Grk2 and Grk6 are most prominently Address correspondence and reprint requests to Dr. John H. Kehrl, Laboratory of expressed in lymphocytes (http://www.immgen.org/databrowser/ Immunoregulation, National Institute of Allergy and Infectious Diseases, National index.html). Linking heterotrimeric G protein signaling to GRK2 Institutes of Health, Building 10, Room 6A01, 10 Center Drive, MSC 1876, regulation, the C-terminal lipid-binding domain in GRK2 (PH Bethesda, MD 20892. E-mail address: [email protected] domain) allows Gbg subunits to recruit GRK2 to the plasma mem- The online version of this article contains supplemental material. brane. In contrast, GRK6 undergoes C-terminal palmitoylation to Abbreviations used in this article: FO, follicular; GPCR, G protein–coupled receptor; GRK, GPCR kinase; HEV, high endothelial venule; KO, knockout; LN, lymph node; mediate membrane localization (11, 12). A limited immune pheno- MZ, marginal zone; S1P, sphingosine-1-phosphate; T, transitional-type; WT, wild-type. typing of Grk6-deficient mice revealed normal B cell chemotaxis to www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900678 2 GRK2-DEFICIENT B CELLS CXCL12 but reduced transendothelial migration (13). Whereas Beads (Thermo Fisher Scientific) individually stained with each fluorochrome. GRK2 deficiency causes embryonic lethality, an analysis of mice Compensation matrices were calculated with FACSDiva software. Data with conditional deletion of Grk2 in B or T lymphocytes has been acquisition was done on FACSCanto II (BD Biosciences) flow cytometer and analyzed with FlowJo software version 9.9.6 (Treestar). reported (14). Follicular (FO) B cells from these mice resisted sphingosine-1-phospate (S1P) R1 desensitization, migrated better S1PR1 immunostaining, internalization, and recovery to S1P in standard chemotaxis assays, but entered lymph nodes For the S1PR1 immunostaining, cells were fixed in 2% paraformaldehyde (LNs) poorly. The S1PR1 receptors on marginal zone (MZ) B cells for 10 min, washed, and stained with S1PR1-PE, which is directed against also resisted desensitization, which impaired MZ B cell shuttling. an external S1PR1 epitope (R & D Systems). To assess S1PR1 internali- In contrast, CXCR4 and CXCR5 signaling was reported as not zation, the splenocytes were rested in DMEM/10 mM HEPES for 30 min at 37˚C/5% CO2 before stimulation with 1 mM S1P or solvent (DMSO) for significantly altered. This study implicated GRK2 as a central 10 or 30 min. Cells were stained with S1PR1-PE prior to stimulation and at regulator of S1PR1 desensitization. the indicated time points. To examine S1PR1 recovery, cells were incu- Building on these results, we have examined in greater detail bated for 10, 30, or 60 min in DMEM/10 mM HEPES at 37˚C/5% CO2. the origins of the phenotypes when B cells lack GRK2. We Cells were washed and stained with S1PR1-PE prior to incubation and at confirmed the finding of impaired S1PR1 desensitization but the indicated times. have also found severe defects in B cell responses to homeostatic