Rap1a Null Mice Have Altered Myeloid Cell Functions Suggesting Distinct Roles for the Closely Related Rap1a and 1b Proteins This information is current as Yu Li, Jingliang Yan, Pradip De, Hua-Chen Chang, Akira of September 25, 2021. Yamauchi, Kent W. Christopherson II, Nivanka C. Paranavitana, Xiaodong Peng, Chaekyun Kim, Veerendra Munugulavadla, Reuben Kapur, Hanying Chen, Weinian Shou, James C. Stone, Mark H. Kaplan, Mary C. Dinauer, Donald L. Durden and Lawrence A. Quilliam Downloaded from J Immunol 2007; 179:8322-8331; ; doi: 10.4049/jimmunol.179.12.8322 http://www.jimmunol.org/content/179/12/8322 http://www.jimmunol.org/ References This article cites 79 articles, 49 of which you can access for free at: http://www.jimmunol.org/content/179/12/8322.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 25, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *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 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Rap1a Null Mice Have Altered Myeloid Cell Functions Suggesting Distinct Roles for the Closely Related Rap1a and 1b Proteins1 Yu Li,2*¶ Jingliang Yan,*¶ Pradip De,ʈ Hua-Chen Chang,†‡§ Akira Yamauchi,§ Kent W. Christopherson II,** Nivanka C. Paranavitana,3*¶ Xiaodong Peng,§ Chaekyun Kim,§# Veerendra Munugulavadla,§ Reuben Kapur,*‡§ Hanying Chen,§ Weinian Shou,*§ James C. Stone,†† Mark H. Kaplan,†‡§ Mary C. Dinauer,†‡§ Donald L. Durden,ʈ and Lawrence A. Quilliam4*¶ The Ras-related GTPases Rap1a and 1b have been implicated in multiple biological events including cell adhesion, free radical Downloaded from production, and cancer. To gain a better understanding of Rap1 function in mammalian physiology, we deleted the Rap1a gene. Although loss of Rap1a expression did not initially affect mouse size or viability, upon backcross into C57BL/6J mice some ,Rap1a؊/؊ embryos died in utero. T cell, B cell, or myeloid cell development was not disrupted in Rap1a ؊/؊ mice. However macrophages from Rap1a null mice exhibited increased haptotaxis on fibronectin and vitronectin matrices that correlated with decreased adhesion. Chemotaxis of lymphoid and myeloid cells in response to CXCL12 or CCL21 was significantly reduced. In contrast, an increase in FcR-mediated phagocytosis was observed. Because Rap1a was previously copurified with the human http://www.jimmunol.org/ neutrophil NADPH oxidase, we addressed whether GTPase loss affected superoxide production. Neutrophils from Rap1a؊/؊ mice had reduced fMLP-stimulated superoxide production as well as a weaker initial response to phorbol ester. These results suggest that, despite 95% amino acid sequence identity, similar intracellular distribution, and broad tissue distribution, Rap1a and 1b are not functionally redundant but rather differentially regulate certain cellular events. The Journal of Immunology, 2007, 179: 8322–8331. he small GTPase Ras is an important regulator of cell but encoded by a separate gene, also exists (7) in addition to three growth and differentiation (1). It operates as a molecular Rap2 proteins that share ϳ65% identity with Rap1 (2, 8, 9). Like switch, alternating between inactive GDP and active Ras, these GTPases are activated by guanine nucleotide exchange by guest on September 25, 2021 T 5 GTP-bound states and is the prototype for more than 130 Ras- factors (GEFs) and turned off by GTPase activating proteins related proteins that regulate a myriad of biological functions (1). (GAPs). The activation of Raps by a large number of diversely Rap1a/Krev-1/Smg p21 was originally identified because of ho- regulated GEFs suggests that they play ubiquitous roles in cell mology with Ras (2), by purification of GTP binding proteins (3, biology triggered by multiple extracellular stimuli and associated 4), by copurification with the neutrophil NADPH oxidase (5), and signaling pathways, reviewed in Ref. 10–12. Although Rap1 and by its ability to revert the phenotype of Ras-transformed cells (6). 2 are regulated by the same GEFs and GAPs, until very recently A closely related protein, Rap1B, sharing 95% amino acid identity little was known about their biological functions. Even less is known about the functional redundancy of Rap1a and 1b. It was originally reported that Rap1 operates as an antagonist of † *Department of Biochemistry and Molecular Biology, Department of Microbiology Ras, competing with it for downstream effectors such as c-Raf-1. and Immunology, ‡Department of Pediatrics, Medical and Molecular Genetics, and §Wells Center for Pediatric Research, Indiana University School of Medicine, Indi- Although this can occur in certain cell types (13), Rap1 has more anapolis, IN 46202; ¶Walther Cancer Institute, Indianapolis, IN 46208; ʈAflac Cancer recently been found to play a number of additional roles. These Center and Blood Disorders, Department of Pediatrics, Emory University, Atlanta, GA 30322; #Inha University College of Medicine, Incheon, Korea; **Rush University include activation of the ERK/MAPK pathway via B-Raf, promo- Medical Center, Chicago, IL 60612; and ††Department of Biochemistry, University of tion of cell-matrix adhesion through inside-out activation of inte- Alberta, Edmonton, Alberta, Canada grins via RAPL and RIAM, and regulation of cell-cell junctions Received for publication June 26, 2007. Accepted for publication October 12, 2007. (reviewed in Refs. 12, 14, 15). Overactivation of Rap1 has also The costs of publication of this article were defrayed in part by the payment of page been recently associated with hemopoietic malignancy (16, 17). charges. This article must therefore be hereby marked advertisement in accordance Loss of the only rap1 gene in Drosophila is embryonic lethal (18). with 18 U.S.C. Section 1734 solely to indicate this fact. Similarly, loss of C3G/RapGEF1, the most ubiquitously expressed 1 This work was supported by an Indiana University School of Medicine Biomedical Research Grant, the Indiana Genomics Initiative of Indiana University, supported in Rap exchange factor is lethal to mice (19, 20). However, because part by Lilly Endowment Incorporated, and by National Institutes of Health Grants C3G also acts on additional Ras family proteins, it was not clear CA108647 and HL69974 (all to L.A.Q.). Y.L. was supported by American Heart Association predoctoral fellowship 0110245Z. 2 Current address: Amgen, One Amgen Center Drive Thousand Oaks, CA 91302. 3 Current address: Applied Biosystems, 2130 Woodward Street, Austin, TX 78744. 5 Abbreviations used in this paper: GEF, guanine nucleotide exchange factor; GAP, 4 Address correspondence and reprint requests to Dr. Lawrence A. Quilliam, Depart- GTPase activating protein; ES cells, embryonic stem cells; KO, knockout; WT, wild ment of Biochemistry and Molecular Biology, 635 Barnhill Drive, MS-4053, Indiana type; MEF, mouse embryo fibroblast; BMD, bone marrow-derived; University School of Medicine, Indianapolis, IN 46202. E-mail address: [email protected] Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org The Journal of Immunology 8323 whether this lethality was solely due to the loss of Rap1 activity fragment as template to confirm the long arm structure and correct inte- (21–24). gration 3Ј to the recombination site. PstI-digested ES cell genomic DNA To better understand the role of Rap1 in mammalian cell biol- was hybridized with a neo probe using a XhoI-SalI fragment from the neomycin resistance gene to confirm correct integration of 5Ј to the re- ogy and to address possible redundancy between the 1a and 1b combination site. Four ES cell clones were confirmed to contain the tar- Ϫ/Ϫ proteins, we generated a mouse lacking the rap1a gene. rap1A geted disruption and used to generate Rap1a KO mice by standard proce- mice initially exhibited no overt phenotype. However, upon back- dures (30) using C57BL/6J blastocysts and pseudopregnant females as crossing mice into the C57BL/6J strain, some embryonic lethality foster mothers. Chimeric mice were successfully generated from just one clone. These were screened by PCR and by Southern blotting for the pres- was observed. Because of the high abundance of Rap1a in human ence of the disrupted Rap1a allele and mated with C57BL/6J mice to platelets (25) and Rap1a in human neutrophils (26) where it asso- produce F1 heterozygous mice. Heterozygotes for the disrupted gene were ciates with the phagocyte NADPH oxidase (5, 27), we examined identified by PCR using digit samples along with three primers, the com- several properties of Rap1a-null white blood cells. Although there mon primer Rap1a 5Ј KO, the neo specific primer ADPR266, and the wild-type (WT) allele specific primer RapExon (5Ј-CCGTAAAATCTGT were no obvious defects in blood cell development, changes in Ј TCTCTC AAGTC-3 ). Confirmed heterozygous F1 mice were mated with macrophage adhesion, haptotaxis,