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dc.contributor.authorDiNitto, Jonathan P.
dc.contributor.authorLee, Meng-Tse
dc.contributor.authorMalaby, Andrew W.
dc.contributor.authorLambright, David G.
dc.date2022-08-11T08:08:00.000
dc.date.accessioned2022-08-23T15:38:44Z
dc.date.available2022-08-23T15:38:44Z
dc.date.issued2010-07-27
dc.date.submitted2010-09-15
dc.identifier.citationBiochemistry. 2010 Jul 27;49(29):6083-92.
dc.identifier.issn1520-4995
dc.identifier.pmid20527794
dc.identifier.urihttp://hdl.handle.net/20.500.14038/25997
dc.description.abstractThe Arf exchange factor Grp1 selectively binds phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P(3)], which is required for recruitment to the plasma membrane in stimulated cells. The mechanisms for phosphoinositide recognition by the PH domain, catalysis of nucleotide exchange by the Sec7 domain, and autoinhibition by elements proximal to the PH domain are well-characterized. The N-terminal heptad repeats in Grp1 have also been shown to mediate homodimerization in vitro as well as heteromeric interactions with heptad repeats in the FERM domain-containing protein Grsp1 both in vitro and in cells [Klarlund, J. K., et al. (2001) J. Biol. Chem. 276, 40065-40070]. Here, we have characterized the oligomeric state of Grsp1 and Grp1 family proteins (Grp1, ARNO, and Cytohesin-1) as well as the oligomeric state, stoichiometry, and specificity of Grsp1 complexes with Grp1, ARNO, and Cytohesin-1. At low micromolar concentrations, Grp1 and ARNO are homodimeric whereas Cytohesin-1 and Grsp1 are monomeric. When mixed with Grsp1, Grp1 homodimers and Cytohesin-1 monomers spontaneously re-equilibrate to form heterodimers, whereas approximately 50% of ARNO remains homodimeric under the same conditions. Fluorescence resonance energy transfer experiments suggest that the Grsp1 heterodimers with Grp1 and Cytohesin-1 adopt a largely antiparallel orientation. Finally, formation of Grsp1-Grp1 heterodimers does not substantially influence the binding of Grp1 to the headgroups of PtdIns(3,4,5)P(3) or PtdIns(4,5)P(2), nor does it influence partitioning with liposomes containing PtdIns(3,4,5)P(3), PtdIns(4,5)P(2), and/or phosphatidylserine.
dc.language.isoen_US
dc.publisherAmerican Chemical Society
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=20527794&dopt=Abstract">Link to article in PubMed</a>
dc.relation.urlhttp://dx.doi.org/10.1021/bi1000454
dc.subjectAmino Acid Sequence
dc.subjectAnimals
dc.subjectCell Membrane
dc.subjectFluorescence Resonance Energy Transfer
dc.subjectGTPase-Activating Proteins
dc.subjectHumans
dc.subjectMice
dc.subjectMolecular Sequence Data
dc.subjectPhosphatidylinositol Phosphates
dc.subjectProtein Multimerization
dc.subjectProtein Structure, Tertiary
dc.subjectReceptors, Cytoplasmic and Nuclear
dc.subjectSignal Transduction
dc.subjectBiochemistry, Biophysics, and Structural Biology
dc.subjectPharmacology, Toxicology and Environmental Health
dc.titleSpecificity and membrane partitioning of Grsp1 signaling complexes with Grp1 family Arf exchange factors
dc.typeJournal Article
dc.source.journaltitleBiochemistry
dc.source.volume49
dc.source.issue29
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/bmp_pp/128
dc.identifier.contextkey1558875
html.description.abstract<p>The Arf exchange factor Grp1 selectively binds phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P(3)], which is required for recruitment to the plasma membrane in stimulated cells. The mechanisms for phosphoinositide recognition by the PH domain, catalysis of nucleotide exchange by the Sec7 domain, and autoinhibition by elements proximal to the PH domain are well-characterized. The N-terminal heptad repeats in Grp1 have also been shown to mediate homodimerization in vitro as well as heteromeric interactions with heptad repeats in the FERM domain-containing protein Grsp1 both in vitro and in cells [Klarlund, J. K., et al. (2001) J. Biol. Chem. 276, 40065-40070]. Here, we have characterized the oligomeric state of Grsp1 and Grp1 family proteins (Grp1, ARNO, and Cytohesin-1) as well as the oligomeric state, stoichiometry, and specificity of Grsp1 complexes with Grp1, ARNO, and Cytohesin-1. At low micromolar concentrations, Grp1 and ARNO are homodimeric whereas Cytohesin-1 and Grsp1 are monomeric. When mixed with Grsp1, Grp1 homodimers and Cytohesin-1 monomers spontaneously re-equilibrate to form heterodimers, whereas approximately 50% of ARNO remains homodimeric under the same conditions. Fluorescence resonance energy transfer experiments suggest that the Grsp1 heterodimers with Grp1 and Cytohesin-1 adopt a largely antiparallel orientation. Finally, formation of Grsp1-Grp1 heterodimers does not substantially influence the binding of Grp1 to the headgroups of PtdIns(3,4,5)P(3) or PtdIns(4,5)P(2), nor does it influence partitioning with liposomes containing PtdIns(3,4,5)P(3), PtdIns(4,5)P(2), and/or phosphatidylserine.</p>
dc.identifier.submissionpathbmp_pp/128
dc.contributor.departmentProgram in Molecular Medicine
dc.contributor.departmentDepartment of Biochemistry and Molecular Pharmacology


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