GTP binding and signaling by G(h)/transglutaminase II involves distinct residues in a unique GTP-binding pocket

G(h) is a dual function protein. It has receptor signaling activity that re quires GTP binding and Ca2+-activated transglutaminase (TGase) activity tha t is inhibited by GTP binding. G(h) shows no homology with other GTP-bindin g proteins, and its GTP-binding site has not been defined. Based on sequenc e analysis of [alpha-P-32]GTP-photolabeled and proteolytically released int ernal peptide fragments, we report localization of GTP binding to a 15-resi due segment ((159)YLTQQGFIYQGSVK(173)) of the Gr, core domain. This was con firmed by site-directed mutagenesis; a G(h)/fXIIIA chimera tin which residu es 162-179 of G(h) were substituted with the equivalent but nonhomologous r egion of the non-GTP-binding TGase factor XIIIA) and a G(h) point mutant, S 171E, retained TGase activity but failed to bind and hydrolyze GTP and did not support alpha(1B)-adrenergic receptor signaling. Slight impairment of G TP binding (1.5-fold) and hydrolysis (10-fold) in the absence of altered TG ase activity did not affect signaling by the mutant K173N, However, greater impairment of GTP binding (g-fold) and hydrolysis (50-fold) abolished sign aling by the mutant H173L, Mutant S171C exhibited enhanced GTP binding and signaling. Thus, residues Ser(171) and Lys(173) are critical for both GTP b inding and signaling but not TGase activity. Mutagenesis of residues N-term inal to Gly(170) impaired both GTP binding and TGase activity. From compute r modeling of G(h), it is evident that the GTP-binding region identified he re is distinct from, but interacts with, the TGase active site. Together wi th structural considerations of G(h) versus other GTP-binding proteins, the se findings indicate that G(h) has a unique GTP-binding pocket and provide for the first time a mechanism for GTP-mediated regulation of the TGase act ivity of G(h).