Muzaddid Sarker1, Ardeshir Goliaei1, Florence Golesi2, Marjorie Poggi2, Aaron A Cook1, Mohammad A I Khan1, Brenda R Temple1,3, Lucia Stefanini4, Matthias Canault2, Wolfgang Bergmeier1,5, Sharon L Campbell1,5. 1. Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. 2. Aix Marseille University, INSERM, INRA, Marseille, France. 3. RL Juliano Structural Bioinformatics Core, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. 4. Department of Internal Medicine and Medical Specialties, Sapienza University of Rome, Rome, Italy. 5. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Abstract
BACKGROUND: The small GTPase Rap1 and its guanine nucleotide exchange factor, CalDAG-GEFI (CDGI), are critical for platelet function and hemostatic plug formation. CDGI function is regulated by a calcium binding EF hand regulatory domain and an atypical C1 domain with unknown function. OBJECTIVE: Here, we investigated whether the C1 domain controls CDGI subcellular localization, both in vitro and in vivo. METHODS: CDGI interaction with phosphoinositides was studied by lipid co-sedimentation assays and molecular dynamics simulations. Cellular localization of CDGI was studied in heterologous cells by immunofluorescence and subcellular fractionation assays. RESULTS: Lipid co-sedimentation studies demonstrated that the CDGI C1 domain associates with membranes through exclusive recognition of phosphoinositides, phosphatidylinositol (4,5)-biphosphate (PIP2) and phosphatidylinositol (3,4,5)-triphosphate (PIP3). Molecular dynamics simulations identified a phospholipid recognition motif consisting of residues exclusive to the CDGI C1 domain. Mutation of those residues abolished co-sedimentation of the C1 domain with lipid vesicles and impaired membrane localization of CDGI in heterologous cells. CONCLUSION: Our studies identify a novel interaction between an atypical C1 domain and phosphatidylinositol (4,5)-biphosphate and phosphatidylinositol (3,4,5)-triphosphate in cellular membranes, which is critical for Rap1 signaling in health and disease.
BACKGROUND: The small GTPase Rap1 and its guanine nucleotide exchange factor, CalDAG-GEFI (CDGI), are critical for platelet function and hemostatic plug formation. CDGI function is regulated by a calcium binding EF hand regulatory domain and an atypical C1 domain with unknown function. OBJECTIVE: Here, we investigated whether the C1 domain controls CDGI subcellular localization, both in vitro and in vivo. METHODS:CDGI interaction with phosphoinositides was studied by lipid co-sedimentation assays and molecular dynamics simulations. Cellular localization of CDGI was studied in heterologous cells by immunofluorescence and subcellular fractionation assays. RESULTS:Lipid co-sedimentation studies demonstrated that the CDGI C1 domain associates with membranes through exclusive recognition of phosphoinositides, phosphatidylinositol (4,5)-biphosphate (PIP2) and phosphatidylinositol (3,4,5)-triphosphate (PIP3). Molecular dynamics simulations identified a phospholipid recognition motif consisting of residues exclusive to the CDGI C1 domain. Mutation of those residues abolished co-sedimentation of the C1 domain with lipid vesicles and impaired membrane localization of CDGI in heterologous cells. CONCLUSION: Our studies identify a novel interaction between an atypical C1 domain and phosphatidylinositol (4,5)-biphosphate and phosphatidylinositol (3,4,5)-triphosphate in cellular membranes, which is critical for Rap1 signaling in health and disease.
Authors: David Van Der Spoel; Erik Lindahl; Berk Hess; Gerrit Groenhof; Alan E Mark; Herman J C Berendsen Journal: J Comput Chem Date: 2005-12 Impact factor: 3.376
Authors: Feng Qian; Guy C Le Breton; Jia Chen; Jing Deng; John W Christman; Dianqing Wu; Richard D Ye Journal: Arterioscler Thromb Vasc Biol Date: 2011-12-29 Impact factor: 8.311
Authors: Luca Monticelli; Senthil K Kandasamy; Xavier Periole; Ronald G Larson; D Peter Tieleman; Siewert-Jan Marrink Journal: J Chem Theory Comput Date: 2008-05 Impact factor: 6.006
Authors: Wenjuan Su; Joseph Wynne; Elaine M Pinheiro; Marianne Strazza; Adam Mor; Emilie Montenont; Jeffrey Berger; David S Paul; Wolfgang Bergmeier; Frank B Gertler; Mark R Philips Journal: Blood Date: 2015-08-31 Impact factor: 22.113
Authors: Agnes Czikora; Daniel J Lundberg; Adelle Abramovitz; Nancy E Lewin; Noemi Kedei; Megan L Peach; Xiaoling Zhou; Raymond C Merritt; Elizabeth A Craft; Derek C Braun; Peter M Blumberg Journal: J Biol Chem Date: 2016-03-28 Impact factor: 5.157
Authors: Brian G Petrich; Patrizia Marchese; Zaverio M Ruggeri; Saskia Spiess; Rachel A M Weichert; Feng Ye; Ralph Tiedt; Radek C Skoda; Susan J Monkley; David R Critchley; Mark H Ginsberg Journal: J Exp Med Date: 2007-12-17 Impact factor: 14.307
Authors: Jill R Crittenden; Shenyu Zhai; Magdalena Sauvage; Takashi Kitsukawa; Eric Burguière; Morgane Thomsen; Hui Zhang; Cinzia Costa; Giuseppina Martella; Veronica Ghiglieri; Barbara Picconi; Karen A Pescatore; Ellen M Unterwald; Walker S Jackson; David E Housman; S Barak Caine; David Sulzer; Paolo Calabresi; Anne C Smith; D James Surmeier; Ann M Graybiel Journal: Neurobiol Dis Date: 2021-08-08 Impact factor: 5.996