title: Nucleobindin 1 is a calcium-regulated guanine nucleotide dissociation inhibitor of G{alpha}i1.
abstract: Nucleobindin 1 (NUCB1) is a widely expressed multidomain calcium-binding protein whose precise physiological and biochemical functions are not well understood. We engineered and heterologously expressed a soluble form of NUCB1 (sNUCB1) and characterized its biophysical and biochemical properties. We show that sNUCB1 exists as a dimer in solution and that each monomer binds two divalent calcium cations. Calcium binding causes conformational changes in sNUCB1 as judged by circular dichroism and fluorescence spectroscopy experiments. Earlier reports suggested that NUCB1 might interact with heterotrimeric G protein α subunits. We show that dimeric calcium-free sNUCB1 binds to expressed Gα(i1) and that calcium binding inhibits the interaction. The binding of sNUCB1 to Gα(i1) inhibits its basal rate of GDP release and slows its rate and extent of GTPγS uptake. Additionally, our tissue culture experiments show that sNUCB1 prevents receptor-mediated Gα(i)-dependent inhibition of adenylyl cyclase. Thus, we conclude that sNUCB1 is a calcium-dependent guanine nucleotide dissociation inhibitor (GDI) for Gα(i1). To our knowledge, sNUCB1 is the first example of a calcium-dependent GDI for heterotrimeric G proteins. We also show that the mechanism of GDI activity of sNUCB1 is unique and does not arise from the consensus GoLoco motif found in RGS proteins. We propose that cytoplasmic NUCB1 might function to regulate heterotrimeric G protein trafficking and G protein-coupled receptor-mediated signal transduction pathways.
authors.affiliation: Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York 10065, USA.
authors.forename: Neeraj
authors.initials: N
authors.lastname: Kapoor
authors.forename: Ruchi
authors.initials: R
authors.lastname: Gupta
authors.forename: Santosh T
authors.initials: ST
authors.lastname: Menon
authors.forename: Ewa
authors.initials: E
authors.lastname: Folta-Stogniew
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Raleigh
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Sakmar
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D011485:Protein Binding; D055503:Protein Multimerization / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D051381:Rats
chemical_list: D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; C000630649:NUCB1 protein, human; D009419:Nerve Tissue Proteins; C000630652:Nucb1 protein, rat; D000080866:Nucleobindins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D002118:Calcium