A Specific Domain of Giα Required for the Transactivation of Giα by Tubulin Is Implicated in the Organization of Cellular Microtubules

Abstract

␣ subunits bind tubulin with high affinity, whereas transducin (G t ␣) does not. The interaction between tubulin and G␣, which also involves the direct transfer of GTP from tubulin to G␣ (transactivation), is not yet fully understood. This study, using chimeras of G i ␣ and G t ␣, showed that the G i ␣ (215-295) segment converted G t ␣ to bind to tubulin and this chimera (chimera 1) could be transactivated by tubulin. Insertion of G t ␣ (237-270) into chimera 1 to form chimera 2 resulted in a protein that, like G t ␣, did not bind tubulin. Thus, it was thought that the G i ␣ (237-270) domain was essential to modulate the binding of G i ␣ 1 to tubulin. Surprisingly, when domain (237-270) of G i ␣ was replaced by G t ␣ (237-270) to form chimera 3, the chimera bound to tubulin with a similar affinity (K D Х120 nM) as wild-type G i ␣ 1 . However, even though chimera 3 displayed normal GTP binding, it was not transactivated by GTP-tubulin. Furthermore, when these chimeras were expressed in COS-1 cells, cellular processes in cells overexpressing G i ␣ 1 or chimera 1 were more abundant and longer than those in native cells. G␣ was seen throughout the length of the process. Morphology of cells expressing chimera 2 was identical to controls. Consistent with the role of Chimera 3 as a "dominant negative" G␣, cells transfected with chimera 3 had only few truncated processes. This study demonstrates that although G i ␣ (237-270) is not obligatory for the binding of G i ␣ to tubulin, it is crucial for the transactivation of G␣ by tubulin. These results also suggest that the transactivation of G␣ by tubulin may play an important role in modulating microtubule organization and cell morphology.G proteins act as intracellular transducers to propagate a variety of signals across the plasma membrane. Due to their interaction with transmembrane receptors and lipid modification, G proteins are usually associated with the plasma membrane. Recently, increasing evidence has emerged that G proteins are also present at intracellular areas such as Golgi apparatus, endoplasmic reticulum, cytoskeleton, and even the nucleus (1-5). Moreover, some studies have also shown that activated G␣s can be released from the plasma membrane to the cytoplasm (6 -9). Thus, it is possible that G proteins exist in several different cellular locations and play roles in various physiologic processes.Microtubules, a major component of the cytoskeleton, are involved in many cellular functions including chromosome movements during mitosis, intracellular transport, and the modulations of cell morphology. The biological function of microtubules is based, in significant part, on the ability of tubulin to polymerize and depolymerize. A heterodimer of ␣-and ␤-tubulin is the basic building block of microtubules. Tubulin is a GTP-binding protein, two GTP molecules are bound noncovalently in exchangeable (E-site on ␤-tubulin) and nonexchangeable (N-site on ␣-tubulin) sites. Both G␣ subunits and tubulin have intrinsic GTPase activity but that of tubulin is activated durin...

Identification and Characterization of GIV, a Novel Gαi/s -interacting Protein Found on COPI, Endoplasmic Reticulum-Golgi Transport Vesicles

“…Yeast two-hybrid 1 to 1 interaction assays of GIV with G␣ family members. GIV interacts with G␣ i1, G␣ 2, G␣ 3, G␣ 0, G␣ z , G s , and the yeast G␣ i homolog GPA1, but it does not interact with G␣ q , G␣ 12 , and G␣ 13 and reacts very weakly with the activated mutant, G␣ s (Q226L). Interactions were analyzed qualitatively by a colony lift assay on three independent clones using 5-bromo-4-chloro-3-indolyl D-galactoside (14), and the appearance of blue colonies was assessed after 2, 4, and 8 h. No background color was detected after 20 h.…”

Activation of Microtubule Dynamics Increases Neuronal Growth via the Nerve Growth Factor (NGF)- and Gαs-mediated Signaling Pathways

“…The full-length constitutively active mutant Q227L G s protein ␣ subunit plasmid was a gift from Dr. Tohru Kozasa (University of Illinois at Chicago). The plasmid encoding NC1 (chimera 3) protein has been described previously (23).…”

“…In addition, we observed that expression of a dominant-negative G␣ i -transducin chimera (NC1) that blocks G␣ s binding to tubulin and G␣ s activation of tubulin GTPase, attenuating microtubule-based cellular projections in COS-1 cells (23). Here NC1 was used to determine the role of G␣ s in neurite outgrowth (Fig.…”

A Molecular and Structural Mechanism for G Protein-mediated Microtubule Destabilization

“…Specifically, the ␣3-␤5 loop of G␣ s was replaced with homologous residues from G␣ t . A similar approach has been used successfully to dissect the interface of G␣ subunits with other proteins, including tubulin (13,16,27,28).…”

“…To further understand the role of these regions in binding, 15-amino acid-long peptides cor- responding to the ␣3-␤5 regions (P3) or residues 28 -42 (peptide N) were synthesized (supplemental Table 2). Control peptides (peptides G t N and G t 3) were derived from G␣ t , which does not bind tubulin (11,27), and corresponded to homologous regions on G␣ s . The affinities of all peptides for tubulin were determined.…”