Tissue transglutaminase (TGase II) is a Ca2+- 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, Gh, is a TGase II that mediates intracellular signaling by the alpha1B-adrenergic receptor (AR) (Nakaoka, H., Perez, D. M., Baek, K. J., Das, T., Husain, A., Mison, K., Im, M.-J., and Graham, R. M. (1994) Science 264, 1593-1596). Here, we evaluated the ability of Gh as compared with Gq to mediate receptor-stimulated inositol phosphate turnover by the three alpha1-subtypes (alpha1A, alpha1B, and alpha1D). In addition, we questioned if the transglutaminase function of Gh 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 (Cys277) 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-->Ser mutant in COS-1 cells. However, like wild-type TGase, the Cys-->Ser mutant mediated receptor-stimulated inositol phosphate turnover when cotransfected with an alpha1B-AR cDNA. Galphaq supported alpha1-AR-mediated inositol phosphate turnover by all three receptor subtypes. By contrast, although both the wild-type and Cys-->Ser construct mediated receptor signaling by the alpha1B AR and alpha1D AR, the alpha1A-AR was unable to interact with Gh. However, a Gh-dependent signaling phenotype could be rescued by a chimeric alpha1A construct in which the third intracellular loop of the alpha1A-AR was replaced by that of the alpha1B-AR. Thus, the signaling function of Gh is independent of its transglutaminase activity and is alpha1-AR subtype specific. This subtype specificity of the interaction between alpha1 ARs and Gh involves important determinants in their third intracellular loops.