Of the 600+ known proteases identified to date in mammals, a significant percentage is involved or implicated in pathogenic and cancer processes. The dipeptidyl peptidase IV (DPIV) gene family, comprising four enzyme members [DPIV (EC 3.4.14.5), fibroblast activation protein, DP8 and DP9] and two nonenzyme members [DP6 (DPL1) and DP10 (DPL2)], are interesting in this regard because of their multiple diverse functions, varying patterns of distribution/localization and subtle, but significant, differences in structure/substrate recognition. In addition, their engagement in cell biological processes involves both enzymatic and nonenzymatic capabilities. This article examines, in detail, our current understanding of the biological involvement of this unique enzyme family and their overall potential as therapeutic targets.

γ-Glutamyl peptides were identified previously as novel positive allosteric modulators of Ca(2+)(o)-dependent intracellular Ca(2+) mobilization in HEK-293 cells that bind in the calcium-sensing receptor VFT domain. In the current study, we investigated whether γ-glutamyl-tripeptides including γ-Glu-Cys-Gly (glutathione) and its analogs S-methylglutathione and S-propylglutathione, or dipeptides including γ-Glu-Ala and γ-Glu-Cys are positive allosteric modulators of Ca(2+)(o)-dependent Ca(2+)(i) mobilization and PTH secretion from normal human parathyroid cells as well as Ca(2+)(o)-dependent suppression of intracellular cAMP levels in calcium-sensing receptor (CaR)-expressing HEK-293 cells. In addition, we compared the effects of the potent γ-glutamyl peptide S-methylglutathione, and the amino acid L-Phe on HEK-293 cells that stably expressed either the wild-type CaR or the double mutant T145A/S170T, which exhibits selectively impaired responses to L-amino acids. We find that γ-glutamyl peptides are potent positive allosteric modulators of the CaR that promote Ca(2+)(o)-dependent Ca(2+)(i) mobilization, suppress intracellular cAMP levels and inhibit PTH secretion from normal human parathyroid cells. Furthermore, we find that the double mutant T145A/S170T exhibits markedly impaired Ca(2+)(i) mobilization and cAMP suppression responses to S-methylglutathione as well as L-Phe indicating that γ-glutamyl peptides and L-amino acids activate the CaR via a common mechanism.

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).

Dipeptidyl peptidase IV (DPPIV) is an atypical serine protease that modifies the biological activities of certain chemokines and neuropeptides. In addition, human DPPIV, also known as the T‐cell activation antigen CD26, binds adenosine deaminase (ADA) to the T‐cell surface, thus protecting the T‐cell from adenosine‐mediated inhibition of proliferation. Mutations were engineered into DPPIV (five point, 16 single point and six deletion mutations) to examine the binding of ADA and 19 monoclonal antibodies. Deletions of C‐terminal residues from the 738‐residue extracellular portion of DPPIV showed that the 214 residues C‐terminal to Ser552 were not required for ADA binding and that peptidase activity could be ablated by deletion of 20 residues from the C‐terminus. Point mutations at either of two locations, Leu294 and Val341, ablated ADA binding. Binding by six anti‐DPPIV antibodies that inhibited ADA binding was found to require Leu340 to Arg343 and Thr440/Lys441 but not the 214 residues C‐terminal to Ser552. The 13 other antibodies studied bound to a truncated DPPIV consisting of amino acids 1–356. Therefore, the binding sites on DPPIV of ADA and antibodies that inhibit ADA binding are discontinuous and overlapping. Moreover, the 47 and 97 residue spacing of amino acids in these binding sites concords with their location on a β propeller fold consisting of repeated β sheets of about 50 amino acids.