Abstract
G(h) is a dual function protein. It has receptor signaling activity that requires GTP binding and Ca(2+)-activated transglutaminase (TGase) activity that is inhibited by GTP binding. G(h) shows no homology with other GTP-binding proteins, and its GTP-binding site has not been defined. Based on sequence analysis of [alpha-(32)P]GTP-photolabeled and proteolytically released internal peptide fragments, we report localization of GTP binding to a 15-residue segment ((159)YVLTQQGFIYQGSVK(173)) of the G(h) core domain. This was confirmed by site-directed mutagenesis; a G(h)/fXIIIA chimera (in which residues 162-179 of G(h) were substituted with the equivalent but nonhomologous region of the non-GTP-binding TGase factor XIIIA) and a G(h) point mutant, S171E, retained TGase activity but failed to bind and hydrolyze GTP and did not support alpha(1B)-adrenergic receptor signaling. Slight impairment of GTP binding (1.5-fold) and hydrolysis (10-fold) in the absence of altered TGase activity did not affect signaling by the mutant K173N. However, greater impairment of GTP binding (6-fold) and hydrolysis (50-fold) abolished signaling by the mutant K173L. Mutant S171C exhibited enhanced GTP binding and signaling. Thus, residues Ser(171) and Lys(173) are critical for both GTP binding and signaling but not TGase activity. Mutagenesis of residues N-terminal to Gly(170) impaired both GTP binding and TGase activity. From computer modeling of G(h), it is evident that the GTP-binding region identified here is distinct from, but interacts with, the TGase active site. Together with structural considerations of G(h) versus other GTP-binding proteins, these 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 activity of G(h).
Highlights
Gh is a dual function protein with transglutaminase (TGase)1 and receptor signaling activities
Together with structural considerations of Gh versus other GTP-binding proteins, these findings indicate that Gh has a unique GTP-binding pocket and provide for the first time a mechanism for GTP-mediated regulation of the TGase activity of Gh
Where Ca2ϩ is present in millimolar concentrations and nucleotides are absent, TGase activity would be stimulated, allowing Gh to function in tissue remodeling
Summary
Constructs—Site-directed mutants were generated in rat Gh cDNA [25]. Overlap polymerase chain reaction was used to construct a Gh/ fXIIIA chimera in which Gh cDNA (positions 480 –542) was substituted with human fXIIIA cDNA (positions 637– 699). WT Gh was further purified (Mono Q® 5/5 column, 4 °C, 50 – 600 mM NaCl linear gradient over 30 min at 0.5 ml/min) and eluted as a single peak at 380 – 420 mM NaCl. Photoaffinity Labeling and Isolation of Radiolabeled Peptide Fragments—WT Gh (10 M) was incubated with 10 M [␣-32P]GTP (3,000 Ci/mmol; NEN Life Science Products) and 100 M GTP (30 °C, 3 h [25], final volume 600 l) and separated from unbound nucleotide (Mono Q® 5/5 column, 4 °C, 50 – 600 mM NaCl gradient). GTP␥S Filter Binding Assays—GTP␥S binding [19] by WT or mutant Gh (6.7 nM) was assessed (20 mM Tris-HCl, pH 7.3, 150 mM NaCl, 1 mM EGTA, 10 mM dithiothreitol, 500 M App(NH)p, 25 nM [35S]GTP␥S (1250 Ci/mmol; NEN Life Science Products), final volume 150 l) with or without 20 M GTP␥S to determine nonspecific and total binding, respectively. Statistical Analyses—All comparisons were done using unpaired Student’s t test. p Ͻ 0.05 was considered significant
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