Abstract
BackgroundHeterotrimeric guanine nucleotide binding proteins of the G12/13 subfamily, which includes the α-subunits Gα12 and Gα13, stimulate the monomeric G protein RhoA through interaction with a distinct subset of Rho-specific guanine nucleotide exchange factors (RhoGEFs). The structural features that mediate interaction between Gα13 and RhoGEFs have been examined in crystallographic studies of the purified complex, whereas a Gα12:RhoGEF complex has not been reported. Several signaling responses and effector interactions appear unique to Gα12 or Gα13, despite their similarity in amino acid sequence.MethodsTo comprehensively examine Gα12 for regions involved in RhoGEF interaction, we screened a panel of Gα12 cassette substitution mutants for binding to leukemia-associated RhoGEF (LARG) and for activation of serum response element mediated transcription.ResultsWe identified several cassette substitutions that disrupt Gα12 binding to LARG and the related p115RhoGEF. These Gα12 mutants also were impaired in activating serum response element mediated signaling, a Rho-dependent response. Most of these mutants matched corresponding regions of Gα13 reported to contact p115RhoGEF, but unexpectedly, several RhoGEF-uncoupling mutations were found within the N- and C-terminal regions of Gα12. Trypsin protection assays revealed several mutants in these regions as retaining conformational activation. In addition, charge substitutions near the Gα12 N-terminus selectively disrupted binding to LARG but not p115RhoGEF.ConclusionsSeveral structural aspects of the Gα12:RhoGEF interface differ from the reported Gα13:RhoGEF complex, particularly determinants within the C-terminal α5 helix and structurally uncharacterized N-terminus of Gα12. Furthermore, key residues at the Gα12 N-terminus may confer selectivity for LARG as a downstream effector.
Highlights
Heterotrimeric guanine nucleotide binding proteins of the G12/13 subfamily, which includes the α-subunits Gα12 and Gα13, stimulate the monomeric G protein RhoA through interaction with a distinct subset of Rho-specific guanine nucleotide exchange factors (RhoGEFs)
Our results suggest a core similarity in the mechanisms utilized by Gα12 and Gα13 to engage RhoGEF targets, it is apparent that several determinants of RhoGEF binding are unique to Gα13
Gα12 and Gα13 define the G12/13 class of heterotrimeric G protein α-subunits, which participate in numerous signaling pathways through stimulation of RhoGEFs that subsequently activate Rho
Summary
Heterotrimeric guanine nucleotide binding proteins of the G12/13 subfamily, which includes the α-subunits Gα12 and Gα13, stimulate the monomeric G protein RhoA through interaction with a distinct subset of Rho-specific guanine nucleotide exchange factors (RhoGEFs). The G12/13 subfamily of heterotrimeric guanine nucleotide binding proteins (G proteins) is comprised of two αsubunits in mammals, Gα12 and Gα13, that have been implicated in a variety of physiological and pathological cellular responses that include proliferation, cytoskeletal rearrangements, migration, and metastatic invasion [1,2]. G12/13 target proteins are a subset of Rho-specific guanine nucleotide exchange factors (RhoGEFs) that activate the monomeric G protein Rho via tandem Dbl-homology /pleckstrin-homology domains [4]. The Rho monomeric GTPases are known primarily for their role in regulating actin cytoskeletal dynamics, but these proteins mediate cell polarity, microtubule dynamics, membrane transport pathways, transcription factor activity, cell growth, and tumorigenesis [5]. Direct interaction with RhoGEFs is required for mutationally activated Gα12 to trigger increased invasiveness of breast cancer cells [9]
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