Characterization of the functional insulin binding epitopes of the full-length insulin receptor

Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1-470) and at the C terminus of the alpha subunit (amino acids 705-715). To evaluate the physiological significance of this ligand binding site, we have transiently expressed cDNAs encoding full-length receptors with alanine mutations of the residues forming the functional epitopes of this binding site and determined their insulin binding properties. Insulin bound to wild-type receptors with complex kinetics, which were fitted to a two-component sequential model; the Kd of the high affinity component was 0.03 nM and that of the low affinity component was 0.4 nM. Mutations of Arg14, Phe64, Phe705, Glu706, Tyr708, Asn711, and Val715 inactivated the receptor. Alanine mutation of Asn15 resulted in a 20-fold decrease in affinity, whereas mutations of Asp12, Gln34, Leu36, Leu37, Leu87, Phe89, Tyr91, Lys121, Leu709, and Phe714 all resulted in 4-10-fold decreases. When the effects of the mutations were compared with those of the same mutations of the secreted recombinant receptor, significant differences were observed for Asn15, Leu37, Asp707, Leu709, Tyr708, Asn711, Phe714, and Val715, suggesting that the molecular basis for the interaction of each form of the receptor with insulin differs. We also examined the effects of alanine mutations of Asn15, Gln34, and Phe89 on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC50 commensurate with their effect on the affinity of the receptor for insulin.     

Comparison of the functional insulin binding epitopes of the A and B isoforms of the insulin receptor

The human insulin receptor is expressed as two isoforms that are generated by alternate splicing of its mRNA; the B isoform has 12 additional amino acids (718-729) encoded by exon 11 of the gene. The isoforms have been reported to have different ligand binding properties. To further characterize their insulin binding properties, we have performed structure-directed alanine-scanning mutagenesis of a major insulin binding site of the receptor, formed from the receptor L1 domain (amino acids 1-470) and amino acids 705-715 at the C terminus of the alpha subunit. Alanine mutants of each isoform were transiently expressed as recombinant secreted extracellular domain in 293 cells, and their insulin binding properties were evaluated by competitive binding assays. Mutation of Arg(86) and Phe(96) of each isoform resulted in receptors that were not secreted. The Kds of unmutated receptors were almost identical for both isoforms. Several new mutations compromising insulin binding were identified. In L1, mutation of Leu(37) decreased affinity 20- to 40-fold and mutations of Val(94), Glu(97), Glu(120), and Lys(121) 3 to 10-fold for each isoform. A number of mutations produced differential effects on the two isoforms. Mutation of Asn(15) in the L1 domain and Phe(714) at the C terminus of the alpha subunit inactivated the A isoform but only reduced the affinity of the B isoform 40- to 60-fold. At the C terminus of the alpha subunit, mutations of Asp(707), Val(713), and Val(715) produced 7- to 16-fold reductions in affinity of the A isoform but were without effect on the B isoform. In contrast, alanine mutations of Tyr(708) and Asn(711) inactivated the B isoform but only reduced the affinities of the A isoform 11- and 6-fold, respectively. In conclusion, alanine-scanning mutagenesis of the insulin receptor A and B isoforms has identified several new side chains contributing to insulin binding and indicates that the energetic contributions of certain side chains differ in each isoform, suggesting that different molecular mechanisms are used to obtain the same affinity.     

Four hydrophobic amino acids of the factor VIII C2 domain are constituents of both the membrane-binding and von Willebrand factor-binding motifs.

Factor VIII binds to phospholipid membranes and to von Willebrand factor (vWf) via its second C domain, which has lectin homology. The crystal structure of the C2 domain has prompted a model in which membrane binding is mediated by two hydrophobic spikes, each composed of a pair of residues displayed on a beta-hairpin turn, and also by net positive charge and specific interactions with phospho-l-serine. To test this model, we prepared 16 factor VIII mutants in which single or multiple amino acids were changed to alanine. Mutants at Arg(2215), Arg(2220), Lys(2227), Lys(2249), Gln(2213), Asn(2217), and Phe(2196)/Thr(2197) had specific activities that were >70% of the wild type. Mutants at Arg(2209), Lys(2227), Trp(2313), and Arg(2320) were degraded within the cell. Hydrophobic spike mutants at Met(2199)/Phe(2200), Leu(2251)/Leu(2252), and Met(2199)/Phe(2200)/Leu(2251)/Leu(2252) (4-Ala) exhibited 43, 59, and 91% reduction in specific activity in the activated partial thromboplastin time assay. In a phospholipid-limiting factor Xa activation assay, these mutants had a 65, 85, and 96% reduction in specific activity. Equilibrium binding of fluorescent, sonicated phospholipid vesicles to mutants immobilized on Superose beads was measured by flow cytometry. The affinities for phospholipid were reduced approximately 20-, 30-, and >35-fold for 2199/2200, 2251/2252, and 4-Ala, respectively. A dimeric form of mature vWf bound to immobilized factor VIII and the same mutants, but the affinities of the mutants were reduced approximately 5-, 10-, and >20-fold, respectively. In a competition, solution phase enzyme-linked immunosorbent assay, plasma vWf bound factor VIII and the same mutants with the affinities for the mutants reduced >5-, >5-, and >50-fold, respectively. We conclude that the two hydrophobic spikes are constituents of both the phospholipid-binding and vWf-binding motifs. In plasma, vWf apparently binds the inherently sticky membrane-binding motif, preventing nonspecific interactions.     

Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. [Phe-1,Val1,Asn2, Gln3,His4,Ser8, His9,Glu12,Tyr15,Leu16]IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has greater than 1,000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. [Gln3,Ala4]IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. [Tyr15,Leu16]IGF-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. This peptide is also equipotent to hIGF-I at the types 1 and 2 IGF receptors. The peptide in which these four-point mutations are combined, [Gln3,Ala4,Tyr15,Leu16]IGF-I, has 600-fold reduced affinity for the serum binding proteins. This peptide has 10-fold increased potency for the insulin receptor, but is equipotent to hIGF-I at the types 1 and 2 IGF receptors. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, these peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I.     

Aromatic residues and neighboring Arg414 in the (6R)-5,6,7, 8-tetrahydro-L-biopterin binding site of full-length neuronal nitric-oxide synthase are crucial in catalysis and heme reduction with NADPH

Nitric-oxide synthase (NOS) requires the cofactor, (6R)-5,6,7, 8-tetrahydrobiopterin (H4B), for catalytic activity. The crystal structures of NOSs indicate that H4B is surrounded by aromatic residues. We have mutated the conserved aromatic acids, Trp(676), Trp(678), Phe(691), His(692), and Tyr(706), together with the neighboring Arg(414) residue within the H4B binding region of full-length neuronal NOS. The W676L, W678L, and F691L mutants had no NO formation activity and had very low heme reduction rates (<0.02 min(-1)) with NADPH. Thus, it appears that Trp(676), Trp(678), and Phe(691) are important to retain the appropriate active site conformation for H4B/l-Arg binding and/or electron transfer to the heme from NADPH. The mutation of Tyr(706) to Leu and Phe decreased the activity down to 13 and 29%, respectively, of that of the wild type together with a dramatically increased EC(50) value for H4B (30-40-fold of wild type). The Tyr(706) phenol group interacts with the heme propionate and Arg(414) amine via hydrogen bonds. The mutation of Arg(414) to Leu and Glu resulted in the total loss of NO formation activity and of the heme reduction with NADPH. Thus, hydrogen bond networks consisting of the heme carboxylate, Tyr(706), and Arg(414) are crucial in stabilizing the appropriate conformation(s) of the heme active site for H4B/l-Arg binding and/or efficient electron transfer to occur.     

Identification of receptor binding and activation determinants in the N-terminal and N-loop regions of the CC chemokine eotaxin

Eotaxin is a CC chemokine that specifically activates the receptor CCR3 causing accumulation of eosinophils in allergic diseases and parasitic infections. Twelve amino acid residues in the N-terminal (residues 1-8) and N-loop (residues 11-20) regions of eotaxin have been individually mutated to alanine, and the ability of the mutants to bind and activate CCR3 has been determined in cell-based assays. The alanine mutants at positions Thr(7), Asn(12), Leu(13), and Leu(20) show near wild type binding affinity and activity. The mutants T8A, N15A, and K17A have near wild type binding affinity for CCR3 but reduced receptor activation. A third class of mutants, S4A, V5A, R16A, and I18A, display significantly perturbed binding affinity for CCR3 while retaining the ability to activate or partially activate the receptor. Finally, the mutant Phe(11) has little detectable activity and 20-fold reduced binding affinity relative to wild type eotaxin, the most dramatic effect observed in both assays but less dramatic than the effect of mutating the corresponding residue in some other chemokines. Taken together, the results indicate that residues contributing to receptor binding affinity and those required for triggering receptor activation are distributed throughout the N-terminal and N-loop regions. This conclusion is in contrast to the separation of binding and activation functions between N-loop and N-terminal regions, respectively, that has been observed previously for some other chemokines.     

Alanine scanning of a putative receptor binding surface of insulin-like growth factor-I

Current evidence supports a binding model in which the insulin molecule contains two binding surfaces, site 1 and site 2, which contact the two halves of the insulin receptor. The interaction of these two surfaces with the insulin receptor results in a high affinity cross-linking of the two receptor alpha subunits and leads to receptor activation. Evidence suggests that insulin-like growth factor-I (IGF-I) may activate the IGF-I receptor in a similar mode. So far IGF-I residues structurally corresponding to the residues of the insulin site 1 together with residues in the C-domain of IGF-I have been found to be important for binding of IGF-I to the IGF-I receptor (e.g. Phe(23), Tyr(24), Tyr(31), Arg(36), Arg(37), Val(44), Tyr(60), and Ala(62)). However, an IGF-I second binding surface similar to site 2 of insulin has not been identified yet. In this study, we have analyzed whether IGF-I residues corresponding to the six residues of the insulin site 2 have a role in high affinity binding of IGF-I to the IGF-I receptor. Six single-substituted IGF-I analogues were produced, each containing an alanine substitution in one of the following positions (corresponding insulin residues in parentheses): Glu(9) (His(B10)), Asp(12) (Glu(B13)), Phe(16) (Leu(B17)), Asp(53) (Ser(A12)), Leu(54) (Leu(A13)), and Glu(58) (Glu(A17)). In addition, two analogues with 2 and 3 combined alanine substitutions were also produced (E9A,D12A IGF-I and E9A,D12A,E58A IGF-I). The results show that introducing alanine in positions Glu(9), Asp(12), Phe(16), Leu(54), and Glu(58) results in a significant reduction in IGF-I receptor binding affinity, whereas alanine substitution at position 53 had no effect on IGF-I receptor binding. The multiple substitutions resulted in a 33-100-fold reduction in IGF-I receptor binding affinity. These data suggest that IGF-I, in addition to the C-domain, uses surfaces similar to those of insulin in contacting its cognate receptor, although the relative contribution of the side chains of homologous residues varies.     

Concerted mutation of Phe residues belonging to the beta-dystroglycan ectodomain strongly inhibits the interaction with alpha-dystroglycan in vitro

The dystroglycan adhesion complex consists of two noncovalently interacting proteins: alpha-dystroglycan, a peripheral extracellular subunit that is extensively glycosylated, and the transmembrane beta-dystroglycan, whose cytosolic tail interacts with dystrophin, thus linking the F-actin cytoskeleton to the extracellular matrix. Dystroglycan is thought to play a crucial role in the stability of the plasmalemma, and forms strong contacts between the extracellular matrix and the cytoskeleton in a wide variety of tissues. Abnormal membrane targeting of dystroglycan subunits and/or their aberrant post-translational modification are often associated with several pathologic conditions, ranging from neuromuscular disorders to carcinomas. A putative functional hotspot of dystroglycan is represented by its intersubunit surface, which is contributed by two amino acid stretches: approximately 30 amino acids of beta-dystroglycan (691-719), and approximately 15 amino acids of alpha-dystroglycan (550-565). Exploiting alanine scanning, we have produced a panel of site-directed mutants of our two consolidated recombinant peptides beta-dystroglycan (654-750), corresponding to the ectodomain of beta-dystroglycan, and alpha-dystroglycan (485-630), spanning the C-terminal domain of alpha-dystroglycan. By solid-phase binding assays and surface plasmon resonance, we have determined the binding affinities of mutated peptides in comparison to those of wild-type alpha-dystroglycan and beta-dystroglycan, and shown the crucial role of two beta-dystroglycan phenylalanines, namely Phe692 and Phe718, for the alpha-beta interaction. Substitution of the alpha-dystroglycan residues Trp551, Phe554 and Asn555 by Ala does not affect the interaction between dystroglycan subunits in vitro. As a preliminary analysis of the possible effects of the aforementioned mutations in vivo, detection through immunofluorescence and western blot of the two dystroglycan subunits was pursued in dystroglycan-transfected 293-Ebna cells.     

Fine-tuning of the binding and dissociation of CO by the amino acids of the heme pocket of Coprinus cinereus peroxidase

Resonance Raman and infrared spectra and the CO dissociation rates (k(off)) were measured in Coprinus cinereus peroxidase (CIP) and several mutants in the heme binding pocket. These mutants included the Asp245Asn, Arg51Leu, Arg51Gln, Arg51Asn, Arg51Lys, Phe54Trp, and Phe54Val mutants. Binding of CO to CIP produced different CO adducts at pH 6 and 10. At pH 6, the bound CO is H-bonded to the protonated distal His55 residue, whereas at alkaline pH, the vibrational signatures and the rate of CO dissociation indicate a distal side which is more open or flexible than in other plant peroxidases. The distal Arg51 residue is important in determining the rate of dissociation in the acid form, increasing by 8-17-fold in the Arg51 mutants compared to that for the wild-type protein. Replacement of the distal Phe with Trp created a new acid form characterized by vibrational frequencies and k(off) values very similar to those of cytochrome c peroxidase.     

Insulin-like growth factor I and its binding proteins: a study of the binding interface using B-domain analogues

The biological activity of the insulin-like growth factors (IGF-I and IGF-II) is regulated by six IGF binding proteins (IGFBPs 1-6). To examine the surface of IGF-I that associates with the IGFBPs, we created a series of six IGF-I analogues, [His(4)]-, [Gln(9)]-, [Lys(9)]-, [Ser(16)]-, [Gln(9),Ser(16)]-, and [Lys(9),Ser(16)]IGF-I, that contained substitutions for residues Thr(4), Glu(9), or Phe(16). Substitution of Ser for Phe(16) did not affect secondary structure but significantly decreased the affinity for all IGFBPs by between 14-fold and >330-fold, indicating that Phe(16) is functionally important for IGFBP association. While His(4) or Gln(9) substitutions had little effect on IGFBP affinity, changing the negative charge of Glu(9) to a positive Lys(9) selectively decreased the affinities of IGFBP-2 and -6 by 140- and 30-fold, respectively. Furthermore, the effects of mutations to both residues 9 and 16 appear to be additive. The analogues are biologically active in rat L6 myoblasts and they retain native structure as assessed by their far-UV circular dichroism (CD) profiles. We propose that Phe(16) and adjacent hydrophobic residues (Leu(5) and Leu(54)) form a functional binding pocket for IGFBP association.     

Delineation of the peptide binding site of the human galanin receptor.

Galanin, a neuroendocrine peptide of 29 amino acids, binds to Gi/Go-coupled receptors to trigger cellular responses. To determine which amino acids of the recently cloned seven-transmembrane domain-type human galanin receptor are involved in the high-affinity binding of the endogenous peptide ligand, we performed a mutagenesis study. Mutation of the His264 or His267 of transmembrane domain VI to alanine, or of Phe282 of transmembrane domain VII to glycine, results in an apparent loss of galanin binding. The substitution of Glu271 to serine in the extracellular loop III of the receptor causes a 12-fold loss in affinity for galanin. We combined the mutagenesis results with data on the pharmacophores (Trp2, Tyr9) of galanin and with molecular modelling of the receptor using bacteriorhodopsin as a model. Based on these studies, we propose a binding site model for the endogenous peptide ligand in the galanin receptor where the N-terminus of galanin hydrogen bonds with Glu271 of the receptor, Trp2 of galanin interacts with the Zn2+ sensitive pair of His264 and His267 of transmembrane domain VI, and Tyr9 of galanin interacts with Phe282 of transmembrane domain VII, while the C-terminus of galanin is pointing towards the N-terminus of th     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

Designing calcium-sensitizing mutations in the regulatory domain of cardiac troponin C

Cardiac troponin C belongs to the EF-hand superfamily of calcium-binding proteins and plays an essential role in the regulation of muscle contraction and relaxation. To follow calcium binding and exchange with the regulatory N-terminal domain (N-domain) of human cardiac troponin C, we substituted Phe at position 27 with Trp, making a fluorescent cardiac troponin C(F27W). Trp(27) accurately reported the kinetics of calcium association and dissociation of the N-domain of cardiac troponin C(F27W). To sensitize the N-domain of cardiac troponin C(F27W) to calcium, we individually substituted the hydrophobic residues Phe(20), Val(44), Met(45), Leu(48), and Met(81) with polar Gln. These mutations were designed to increase the calcium affinity of the N-domain of cardiac troponin C by facilitating the movement of helices B and C (BC unit) away from helices N, A, and D (NAD unit). As anticipated, these selected hydrophobic residue substitutions increased the calcium affinity of the regulatory domain of cardiac troponin C(F27W) approximately 2.1-15.2-fold. Surprisingly, the increased calcium affinity caused by the hydrophobic residue substitutions was largely due to faster calcium association rates (2.6-8.7-fold faster) rather than to slower calcium dissociation rates (1.2-2.9-fold slower). The regulatory N-domains of cardiac troponin C(F27W) and its mutants were also able to bind magnesium competitively and with physiologically relevant affinities (1.2-2.7 mm). The design of calcium-sensitizing cardiac troponin C mutants presented in this work enhances the understanding of how to control cation binding properties of EF-hand proteins and ultimately their structure and physiological function.     

Structure-function analysis of the DNA binding domain of Saccharomyces cerevisiae ABF1.

To localize the DNA binding domain of the Saccharomyces cerevisiae Ars binding factor 1 (ABF1), a multifunctional DNA binding protein, plasmid constructs carrying point mutations and internal deletions in the ABF1 gene were generated and expressed in Escherichia coli. Normal and mutant ABF1 proteins were purified by affinity chromatography and their DNA binding activities were analyzed. The substitution of His61, Cys66 and His67 respectively, located in the zinc finger motif in the N-terminal region (amino acids 40-91), eliminated the DNA binding activity of ABF1 protein. Point mutations in the middle region of ABF1, specifically at Leu353, Leu399, Tyr403, Gly404, Phe410 and Lys434, also eliminated or reduced DNA binding activity. However, the DNA binding activity of point mutants of Ser307, Ser496 and Glu649 was the same as that of wild-type ABF1 protein and deletion mutants of amino acids 200-265, between the zinc finger region and the middle region (residues 323-496) retained DNA binding activity. As a result, we confirmed that the DNA binding domain of ABF1 appears to be bipartite and another DNA binding motif, other than the zinc finger motif, is situated between amino acid residues 323 and 496.     

A hydrophobic sequence at position 313-316 (Leu-Ala-Phe-Trp) in the fifth domain of apolipoprotein H (beta2-glycoprotein I) is crucial for cardiolipin binding.

Apolipoprotein H (apoH, protein; APOH, gene) binds to negatively charged phospholipids, which triggers the production of a subset of autoantibodies against phospholipid in patients with autoimmune diseases. We have demonstrated that two naturally occurring missense mutations in the fifth domain of apoH, Trp316Ser and Cys306Gly, disrupt the binding of native apoH to phosphatidylserine [Sanghera, D. K., Wagenknecht, D. R., McIntyre, J. A. & Kamboh, M. I. (1997) Hum. Mol. Genet. 6, 311-316]. To confirm whether these are functional mutations, we mutagenized APOH cDNAs and transiently expressed them in COS-1 cells. The cardiolipin ELISA of wild-type and mutant recombinant apoH confirmed that the Gly306 and Ser316 mutations are responsible for abolishing the binding of recombinant apoH to cardiolipin. These mutations, however, had no effect on the levels of expression or secretion of recombinant apoH in transfected COS-1 cells. While the Cys306Gly mutation disrupts a disulfide bond between Cys306 and Cys281, which appears to be critical for clustering positively charged amino acids, the Trp316Ser mutation affects the integrity of an evolutionarily conserved hydrophobic sequence at position 313-316 (Leu-Ala-Phe-Trp), which is hypothesized to interact with anionic phospholipid. To test this hypothesis, we exchanged the remaining three hydrophobic amino acids with neutral amino acids by site-directed mutagenesis (Leu313Gly, Ala314Ser and Phe315Ser). Binding of the Leu313Gly and Phe315Ser mutants to cardiolipin was significantly reduced to 25% and 13%, respectively, of that of the wild-type. On the other hand, the Ala314Ser mutation showed normal cardiolipin binding. Taken together with our previous findings, these results strongly suggest that the configuration of the fifth domain of apoH, as well as the integrity of the highly conserved hydrophobic amino acids at positions 313-316, is essential for the binding of apoH to anionic phospholipid.     

Protein kinase C contains two phorbol ester binding domains

A series of deletion and truncation mutants of protein kinase C (PKC) were expressed in the baculovirus-insect cell expression system in order to elucidate the ability of various domains of the enzyme to bind phorbol dibutyrate (PDBu). A PKC truncation mutant consisting of only the catalytic domain of the enzyme did not bind [3H]PDBu, whereas a PKC truncation mutant consisting of the regulatory domain (containing the tandem cysteine-rich putative zinc finger regions) bound [3H]PDBu. Deletion of the second conserved region (C2) of PKC did not abolish [3H]PDBu binding, whereas a deletion of the first conserved region (C1) of PKC, containing the two cysteine-rich sequences, completely abolished [3H]PDBu binding. Additional truncation and deletion mutants helped to localize the region necessary for [3H]PDBu binding; all PKC mutants that contained either one of the cysteine-rich zinc finger-like regions possessed phorbol ester binding activity. Scatchard analyses of these mutants indicated that each bound [3H]PDBu with equivalent affinity (21-41 nM); approximately 10-20-fold less than the native enzyme. In addition, a peptide of 146 amino acid residues from the first cysteine-rich region, as well as a peptide of only 86 amino acids residues from the second cysteine-rich region, both bound [3H]PDBu with high affinity (31 +/- 4 and 59 +/- 13 nM, respectively). These data establish that PKC contains two phorbol ester binding domains which may function in its regulation.     

Kinetic properties of aromatase mutants Pro308Phe, Asp309Asn, and Asp309Ala and their interactions with aromatase inhibitors.

Mutant forms of aromatase cytochrome P-450 bearing modifications of amino acid residues Pro308 and Asp309 and expressed in transfected Chinese hamster ovary cells were subjected to kinetic analysis and inhibition studies. The Km for androstenedione for expressed wild type (11.0 +/- 0.3 nM SEM, n = 3) increased 4-, 25- and 31-fold for mutants Pro308Phe, Asp309Asn and Asp309Ala, respectively. There were significant differences in sensitivity among wild type and mutants to highly selective inhibitors of estrogen biosynthesis. 4-Hydroxyandrostenedione (4-OHA) a strong inhibitor of wild type aromatase activity (IC50 = 21 nM and Ki = 10 nM), was even more effective against mutant Pro308Phe (IC50 = 13 nM and Ki = 2.8 nM), but inhibition of mutants Asp309Asn and Asp309Ala was considerably less (IC50 = 345 and 330 nM and Ki = 55 and 79 nM, respectively). Expressed wild type aromatase and Pro308Phe aromatase were strongly inhibited by CGS 16949A (IC50 = 4.0 and 4.6 nM, respectively) whereas mutants Asp309Asn and Asp309Ala were markedly less sensitive (IC50 = 140 and 150 nM, respectively). CGS 18320B produced similar inhibition. Kinetic analyses produced Ki = 0.4 nM for CGS 16949A inhibition of wild type versus 1.1, 37 and 58 nM, respectively, against Pro308Phe, Asp309Asn and Asp309Ala. The results demonstrate significant changes in function resulting from single amino acid modifications of the aromatase enzyme. Our data indicate that mutation in Asp309 creates a major distortion in the substrate binding site, rendering the enzyme much less efficient for androstenedione aromatization. The substitution of Pro308 with Phe produces weaker affinity for androstenedione in the substrate pocket, but this alteration favors 4-OHA binding. Similarly, mutant Pro308Phe exhibits a slightly greater sensitivity to inhibition by CGS 18320B than does the wild type. These results indicate that residues Pro308 and Asp309 play critical roles in determining substrate specificity and catalytic capability in aromatase.     

Disease-causing mutations in cardiac troponin T: identification of a critical tropomyosin-binding region

Fifteen percent of the mutations causing familial hypertrophic cardiomyopathy are in the troponin T gene. Most mutations are clustered between residues 79 and 179, a region known to bind to tropomyosin at the C-terminus near the complex between the N- and C-termini. Nine mutations were introduced into a troponin T fragment, Gly-hcTnT(70-170), that is soluble, alpha-helical, binds to tropomyosin, promotes the binding of tropomyosin to actin, and stabilizes an overlap complex of N-terminal and C-terminal tropomyosin peptides. Mutations between residues 92 and 110 (Arg92Leu, Arg92Gln, Arg92Trp, Arg94Leu, Ala104Val, and Phe110Ile) impair tropomyosin-dependent functions of troponin T. Except for Ala104Val, these mutants bound less strongly to a tropomyosin affinity column and were less able to stabilize the TM overlap complex, effects that were correlated with increased stability of the troponin T, measured using circular dichroism. All were less effective in promoting the binding of tropomyosin to actin. Mutations within residues 92-110 may cause disease because of altered interaction with tropomyosin at the overlap region, critical for cooperative actin binding and regulatory function. A model for a five-chained coiled-coil for troponin T in the tropomyosin overlap complex is presented. Mutations outside the region (Ile79Asn, Delta 160Glu, and Glu163Lys) functioned normally and must cause disease by another mechanism.     

癌肿与氨基酸代谢的研究

研究了癌肿与氨基酸代谢的关系。这些癌肿包括喉癌HepⅡ细胞 ,急性非淋巴细胞白血病和急性淋巴细胞白血病 ,结果表明 :( 1 )喉癌细胞株培养过程中亮氨酸、赖氨酸、丝氨酸、天冬酰胺、异亮氨酸、甘氨酸以及苏氨酸等水平明显降低 ,而色氨酸水平明显增加 ,说明喉癌细胞的生长繁殖必须依赖以上 7种氨基酸同时释放了色氨酸 ;( 2 )急性非淋巴细胞白血病 (ANLL)患者血浆中的谷氨酸、甘氨酸、亮氨酸、苯丙氨酸、酪氨酸和色氨酸等水平明显升高 ,而苏氨酸、组氨酸、丙氨酸等水平明显降低 ,这些结果与国际报道相一致 ;( 3)经治疗后 ,ANLL患者血浆中甘氨酸、色氨酸和苯丙氨酸等水平明显降低 ,而丙氨酸、组氨酸等水平明显升高 ,表明肿瘤细胞处在无氧代谢。患者经治疗后色氨酸和苯丙氨酸水平降低和组氨酸水平的升高对患者预后是有益的 ;( 4)急性淋巴细胞白血病患者血浆中苯丙氨酸、赖氨酸、色氨酸和酪氨酸水平提高 ,这些氨基酸能促进肿瘤生长 ,而门冬酰胺、谷氨酰胺以及天冬氨酸水平降低 ,说明这 3种氨基酸为肿瘤生长所必须。此外还发现ALL患者外周淋巴细胞中精氨酸水平增加 ,精氨酸对癌肿细胞有直接杀伤作用。     

Role of insulin receptor dimerization domains in ligand binding, cooperativity, and modulation by anti-receptor antibodies

To define the structures within the insulin receptor (IR) that are required for high affinity ligand binding, we have used IR fragments consisting of four amino-terminal domains (L1, cysteine-rich, L2, first fibronectin type III domain) fused to sequences encoded by exon 10 (including the carboxyl terminus of the alpha-subunit). The fragments contained one or both cysteine residues (amino acids 524 and 682) that form disulfides between alpha-subunits in native IR. A dimeric fragment designated IR593.CT (amino acids 1-593 and 704-719) bound (125)I-insulin with high affinity comparable to detergent-solubilized wild type IR and mIR.Fn0/Ex10 (amino acids 1-601 and 650-719) and greater than that of dimeric mIR.Fn0 (amino acids 1-601 and 704-719) and monomeric IR473.CT (amino acids 1-473 and 704-719). However, neither IR593.CT nor mIR.Fn0 exhibited negative cooperativity (a feature characteristic of the native insulin receptor and mIR.Fn0/Ex10), as shown by failure of unlabeled insulin to accelerate dissociation of bound (125)I-insulin. Anti-receptor monoclonal antibodies that recognize epitopes in the first fibronectin type III domain (amino acids 471-593) and inhibit insulin binding to wild type IR inhibited insulin binding to mIR.Fn0/Ex10 but not IR593.CT or mIR.Fn0. We conclude the following: 1) precise positioning of the carboxyl-terminal sequence can be a critical determinant of binding affinity; 2) dimerization via the first fibronectin domain alone can contribute to high affinity ligand binding; and 3) the second dimerization domain encoded by exon 10 is required for ligand cooperativity and modulation by antibodies.