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.
Highlights
From the ‡Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York 10065, the §W
We show that the mechanism of guanine nucleotide dissociation inhibitor (GDI) activity of soluble form of NUCB1 (sNUCB1) is unique and does not arise from the consensus GoLoco motif found in RGS proteins
This lifetime is regulated by GTPase-accelerating proteins (GAPs), which catalyze the rapid hydrolysis of the G␣-bound GTP to GDP and by guanine nucleotide dissociation inhibitors (GDIs), which inhibit the exchange of GDP for GTP in the catalytic pocket of G␣ [6]
Summary
Thereafter, the intrinsic GTPase activity of G␣ reverts it back to the GDP-bound state, which can reassociate with G␥ This inhibits the interaction of G protein subunits with downstream effectors, which results in the turning-off of the signaling pathways. In 1999, Lanier and co-workers [11] used yeast two-hybrid analysis to identify distinct receptor-independent activators of G protein signaling or AGS proteins Several members of this AGS family (AGS3-6) have been shown to function as GDIs of G␣i subunits [12]. We present the detailed biophysical and biochemical characterization of the Ca2ϩ-binding ability and the oligomeric state of a heterologously expressed N-terminally truncated soluble form of NUCB1, termed sNUCB1 (Fig. 1A). Our findings suggest that NUCB1 is involved in regulating cytoplasmic G protein signaling pathways
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