Dipeptidyl peptidase IV (DPP-IV/CD26) is a multifunctional type II transmembrane serine peptidase. This enzyme contributes to the regulation of various physiological processes, including blood sugar homeostasis, by cleaving peptide hormones, chemokines and neuropeptides. We have determined the 2.5 A structure of the extracellular region of DPP-IV in complex with the inhibitor valine-pyrrolidide. The catalytic site is located in a large cavity formed between the alpha/beta-hydrolase domain and an eight-bladed beta-propeller domain. Both domains participate in inhibitor binding. The structure indicates how substrate specificity is achieved and reveals a new and unexpected opening to the active site.

Alanine scanning mutagenesis has been used to identify specific side chains of insulin which strongly influence binding to the insulin receptor. A total of 21 new insulin analog constructs were made, and in addition 7 high pressure liquid chromatography-purified analogs were tested, covering alanine substitutions in positions B1, B2, B3, B4, B8, B9, B10, B11, B12, B13, B16, B17, B18, B20, B21, B22, B26, A4, A8, A9, A12, A13, A14, A15, A16, A17, A19, and A21. Binding data on the analogs revealed that the alanine mutations that were most disruptive for binding were at positions TyrA19, GlyB8, LeuB11, and GluB13, resulting in decreases in affinity of 1,000-, 33-, 14-, and 8-fold, respectively, relative to wild-type insulin. In contrast, alanine substitutions at positions GlyB20, ArgB22, and SerA9 resulted in an increase in affinity for the insulin receptor. The most striking finding is that B20Ala insulin retains high affinity binding to the receptor. GlyB20 is conserved in insulins from different species, and in the structure of the B-chain it appears to be essential for the shift from the ␣-helix B8 -B19 to the ␤-turn B20 -B22. Thus, replacing GlyB20 with alanine most likely modifies the structure of the B-chain in this region, but this structural change appears to enhance binding to the insulin receptor.Insulin mediates its effects by binding to the insulin receptor in the plasma membrane of target cells. The molecular mechanisms for insulin interaction with the receptor are not fully understood. The crystal structure of the insulin molecule has been known for more than 25 years (1), but it remains an open question whether the structure of insulin that binds to the receptor is similar to the crystal structure. Until the structure of bound insulin and the side chains that are actually involved in binding is identified by co-crystallization of the receptor and ligand, more information about the binding domain on insulin can be obtained using mutational approaches.The binding domain of the insulin molecule has been studied by investigating receptor binding of a number of insulins from different animal species as well as chemically modified and more recently genetically engineered insulins (2-4). These studies have provided experimental support for a model in which invariant residues from both A and B chains form a surface that binds to the insulin receptor. The putative binding domain comprises a number of residues overlapping the dimerforming surface (ValB12, TyrB16, GlyB23, PheB24, PheB25, TyrB26, GlyA1, GlnA5, TyrA19, and AsnA21) and some of the residues buried beneath the COOH terminus of the B-chain (IleA2, ValA3, GluA4) (2). Cross-linking studies with an azidophenylalanine-substituted analog have shown that one of these residues, PheB25, comes into close proximity to the insulin receptor upon binding (5).Recently, a second binding site has been proposed, involving residues LeuA13 and LeuB17 (6, 7). A biphasic binding reaction involving this second binding site could explain the negative cooperativity phenomen...

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Binding of insulin to its receptor (IR) causes rapid autophosphorylation with concomitant activation of its tyrosine kinase which transmits the signal by phosphorylating cellular substrates. The IR activity is controlled by protein-tyrosine phosphatases, but those directly involved in regulating the insulin receptor and its signaling pathways have not yet been identified. Using baby hamster kidney cells overexpressing the IR and a novel insulin-based selection principle, we established stable cell lines with functionally coupled expression of the IR and protein-tyrosine phosphatases. The two closely related protein-tyrosine phosphatases ␣ and ⑀ were identified as negative regulators of IR tyrosine kinase.Insulin is an important regulator of different metabolic processes and plays a key role in the control of blood glucose. Defects related to its synthesis or signaling lead to diabetes mellitus. Binding of insulin to its receptor causes rapid autophosphorylation of several tyrosine residues in the intracellular part of the ␤-subunit. Three closely positioned tyrosine residues (the tyrosine 1150 domain) must all be phosphorylated to obtain full activity of the insulin receptor tyrosine kinase (IRTK) 1 which transmits the signal further downstream by tyrosine phosphorylation of other cellular substrates, including insulin receptor substrate-1 (IRS-1) (1-4). The structural basis for the function of the tyrosine triplet has been provided by recent x-ray crystallographic studies of IRTK that showed tyrosine 1150 to be autoinhibitory in its unphosphorylated state (5).Several studies clearly indicate that the activity of the autophosphorylated IRTK can be reversed by dephosphorylation in vitro (reviewed in Goldstein (6)) (7,8), with the triphosphorylated tyrosine 1150 domain being the most sensitive target for protein-tyrosine phosphatases (PTPs) as compared to the diand monophosphorylated forms (8). It is, therefore, tempting to speculate that this tyrosine triplet functions as a control switch of IRTK activity. Indeed, the IRTK appears to be tightly regulated by PTP-mediated dephosphorylation in vivo (9 -11). The intimate coupling of PTPs to the insulin signaling pathway is further evidenced by the finding that insulin differentially regulates PTP activity in rat hepatoma cells (12) and in livers from alloxan diabetic rats (13). However, little is known about the identity of the PTPs involved in IRTK regulation.To identify PTPs that negatively regulate the IRTK activity we developed a novel selection principle that allows establishment of stable cell lines with functionally coupled overexpression of IR and inhibitory PTPs. For this purpose we used a previously established baby hamster kidney cell line (BHK-IR) (14), which exhibits high levels of IR expression and responds to insulin stimulation with complete growth inhibition of adherent cells. PTPs that impede or block the insulin signal can consequently be identified by their capacity to restore cell growth. This effect was found to be induced through direct activ...