alpha sub(1)-Adrenergic receptor signaling via G sub(h) is subtype specific and independent of its transglutaminase activity

Tissue transglutaminase (TGase II) is a Ca super(2+)- and thiol-dependent enzyme that catalyzes the post-translational modification of proteins via the formation of epsilon ( gamma -glutamyl)lysine bonds. We have shown previously that the GTP-binding protein, G sub(h), is a TGase II that mediates intracellular signaling by the alpha sub(1B)-adrenergic receptor (AR). Here, we evaluated the ability of G sub(h) as compared with G sub(q) to mediate receptor-stimulated inositol phosphate turnover by the three alpha sub(1)-subtypes ( alpha sub(1A), alpha sub(1B), and alpha sub(1D)). In addition, we questioned if the transglutaminase function of G sub(h) is involved in its receptor signaling activity. A mutant form of a human TGase II cDNA in which the codon for the active site cysteine (Cys super(277)) was replaced by serine was cloned into the mammalian expression vector pMT2'. Compared with wild-type TGase II, no transglutaminase activity was observed with transient transfection of this Cys arrow right Ser mutant in COS-1 cells. However, like wild-type TGase, the Cys arrow right Ser mutant mediated receptor-stimulated inositol phosphate turnover when cotransfected with an alpha sub(1B)-AR cDNA. G sub( alpha q) supported alpha sub(1)-AR-mediated inositol phosphate turnover by all three receptor subtypes. By contrast, although both the wild-type and Cys arrow right Ser construct mediated receptor signaling by the alpha sub(1B) AR and alpha sub(1D) AR, the alpha sub(1A)-AR was unable to interact with G sub(h). However, a G sub(h)-dependent signaling phenotype could be rescued by a chimeric alpha sub(1A) construct in which the third intracellular loop of the alpha sub(1A)-AR was replaced by that of the alpha sub(1B)-AR. Thus, the signaling function of G sub(h) is independent of its transglutaminase activity and is alpha sub(1)-AR subtype specific. This subtype specificity of the interaction between alpha sub(1) ARs and G sub(h) involves important determinants in their third intracellular loops.