Specificity and membrane partitioning of Grsp1 signaling complexes with Grp1 family Arf exchange factors.
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.
Program in Molecular Medicine and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Jonathan P
JP
DiNitto
Meng-Tse
MT
Lee
Andrew W
AW
Malaby
David G
DG
Lambright
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D020690:GTPase-Activating Proteins; D018129:Phosphatidylinositol Phosphates; D018160:Receptors, Cytoplasmic and Nuclear; C102474:cytohesin-2; C060974:phosphatidylinositol 3,4,5-triphosphate; C040985:phosphatidylinositol receptors