Heterotrimeric G proteins are molecular switches modulated by families of structurally and functionally related regulators. GIV (G␣-interacting vesicle-associated protein) is the first nonreceptor guanine nucleotide exchange factor (GEF) that activates G␣ i subunits via a defined, evolutionarily conserved motif. Here we found that Calnuc and NUCB2, two highly homologous calcium-binding proteins, share a common motif with GIV for G␣ i binding and activation. Bioinformatics searches and structural analysis revealed that Calnuc and NUCB2 possess an evolutionarily conserved motif with sequence and structural similarity to the GEF sequence of GIV. Using in vitro pulldown and competition assays, we demonstrate that this motif binds preferentially to the inactive conformation of G␣ i1 and G␣ i3 over other G␣ subunits and, like GIV, docks onto the ␣3/switch II cleft. Calnuc binding was impaired when Lys-248 in the ␣3 helix of G␣ i3 was replaced with M, the corresponding residue in G␣ o , which does not bind to Calnuc. Moreover, mutation of hydrophobic residues in the conserved motif predicted to dock on the ␣3/switch II cleft of G␣ i3 impaired the ability of Calnuc and NUCB2 to bind and activate G␣ i3 in vitro. We also provide evidence that calcium binding to Calnuc and NUCB2 abolishes their interaction with G␣ i3 in vitro and in cells, probably by inducing a conformational change that renders the G␣ i -binding residues inaccessible. Taken together, our results identify a new type of G␣ i -regulatory motif named the GBA motif (for G␣-binding and -activating motif), which is conserved across different proteins throughout evolution. These findings provide the structural basis for the properties of Calnuc and NUCB2 binding to G␣ subunits and its regulation by calcium ions.

Heterotrimeric G proteins are critical signal-transducing molecules controlled by a complex network of regulators. GIV (a.k.a. Girdin) is a unique component of this network and a nonreceptor guanine nucleotide exchange factor (GEF) that functions via a signature motif. GIV's GEF motif is involved in the regulation of critical biological processes such as phosphoinositide 3 kinase (PI3K)-Akt signaling, actin cytoskeleton remodeling, cell migration, and cancer metastasis. Here we investigated how the GEF function of GIV affects the wiring of its signaling pathway to shape different biological responses. Using a structure-guided approach, we designed a battery of GIV mutants with different Gαi-binding and -activating properties and used it to dissect the specific impact of changes in GIV's GEF activity on several cellular responses. In vivo signaling assays revealed a threshold effect of GEF activity for the activation of Akt by GIV in different cell lines and by different stimuli. Akt signaling is minimal at low GEF activity and is sharply increased to reach a maximum above a threshold of GEF activity, suggesting that GIV is a critical signal amplifier and that activation of Akt is ultrasensitive to changes in GIV's GEF activity. A similar threshold dependence was observed for other biological functions promoted by GIV such as remodeling of the actin cytoskeleton and cell migration. This functional characterization of GIV's GEF motif provides insights into the molecular interactions between nonreceptor GEFs and G proteins and the mechanisms that govern this signal transduction pathway.

In this case report, we describe a 55-year-old female patient with worsening exertional dyspnea who is referred to the cardiology department, due to the appearance of worsening pulmonary vascular disease on computed tomography (CT) of the chest. Previous transthoracic echocardiograms (TTE) identified right ventricle enlargement, but no other structural abnormalities. She completed cardiac magnetic resonance (CMR) imaging, which identified a large secundum atrial septal defect (ASD). She subsequently underwent surgical planning and correction of the lesion with improvement of her symptoms. This case and a growing body of literature support the use of CMR as an alternative imaging modality for the diagnosis of congenital heart disease (CHD).

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Heterotrimeric G proteins are molecular switches modulated by families of structurally and functionally related regulators. GIV is the first non‐receptor GEF that activates Gαi‐subunits via a defined, evolutionarily conserved motif. Here we found that Calnuc and its closely related homolog NUCB2, two calcium‐binding proteins, share a common motif with GIV for Gαi binding and activation. Bioinformatic searches and structural analysis revealed that Calnuc and NUCB2 possess an evolutionarily conserved motif with sequence and structural similarity to GIV's GEF sequence. Using in vitro pulldown and competition assays we demonstrate that this motif binds preferentially to the inactive conformation of Gαi1 and Gαi3 over other Gα‐subunits and docks onto the same site as GIV, i.e., the α3/Switch II cleft. Calnuc binding was impaired when K248 in the α3 helix of Gαi3 was replaced by the corresponding M residue in Gαo, which does not bind to Calnuc. Moreover, mutation of hydrophobic residues from the conserved motif predicted to dock on the α3/Switch II cleft of Gαi3 abolished Calnuc's binding to Gαi3 and impaired its GEF activity in vitro. We also demonstrate that calcium binding to Calnuc and NUCB2 abolishes their interaction with Gαi3, probably via a conformational change that renders their Gαi‐binding residues unexposed. Taken together our results define a new class of structurally‐related Gαi regulatory motifs and provide the structural basis for the properties of Calnuc and NUCB2 binding to Gα‐subunits and its regulation by calcium ions.Support: NIH grants DKI7780 and CA100768 to M.G.F. Susan G. Komen fellowship KG080079 to M.G‐M CAMS (BWF) and RSA (AGA) to P.G.

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