Association between the PDGF receptor and members of the src family of tyrosine kinases.

We have examined the interaction between the platelet-derived growth factor (PDGF) receptor and three src family tyrosine kinases, pp60c-src, p59fyn, and pp62c-yes. The kinase activities of all three enzymes were elevated after PDGF stimulation of quiescent fibroblasts, coincident with association of the src family kinases with the PDGF receptor and other proteins. The presence of a protein of 81-85 kd in these complexes correlated with the detection of phosphatidylinositol (PI) kinase activity (previously described to associate with both the PDGF receptor and pp60c-src-middle T antigen). These results suggest that the physiological response to PDGF involves interaction of the receptor not only with serine/threonine and lipid kinases and a phospholipase, but also with other tyrosine kinases.     

A single amino acid substitution in a hydrophobic domain causes temperature-sensitive cell-surface transport of a mutant viral glycoprotein.

DNA sequences were determined for three cDNA clones encoding vesicular stomatitis virus glycoproteins from the tsO45 mutant (which encodes a glycoprotein that exhibits temperature-sensitive cell-surface transport), the wild-type parent strain, and a spontaneous revertant of tsO45. The DNA sequence analysis showed that as many as three amino acid changes could be responsible for the transport defect. By recombining the cDNA clones in vitro and expressing the recombinants in COS cells, we were able to trace the critical lesion in tsO45 to a single substitution of a polar amino acid (serine) for a hydrophobic amino acid (phenylalanine) in a hydrophobic domain. We suggest that this nonconservative substitution may block protein transport by causing protein denaturation at the nonpermissive temperature. Comparison of the predicted glycoprotein sequences from two vesicular stomatitis virus strains suggests a possible basis for the differential carbohydrate requirement in transport of the two glycoproteins.     

Activation of insulin receptor signaling by a single amino acid substitution in the transmembrane domain.

The insulin receptor is a ligand-activated tyrosine kinase composed of two alpha and two beta subunits. A single transmembrane domain composed of 23 hydrophobic residues is contained in each beta subunit. We examined the role of the transmembrane domain in regulating insulin receptor signaling by inserting a negatively charged amino acid (Asp) for Val938 (V938D). Chinese hamster ovary (CHO) cells were stably transfected with a plasmid containing both the neomycin-resistance gene and either the wild-type or the mutant (V938D) insulin receptor cDNA. Insulin binding increased similarly in CHO cells stably transfected with the wild-type and the V938D-mutant insulin receptor cDNA. Insulin stimulated glucose transport and cell growth in cells expressing the normal insulin receptor. By contrast, in the absence of insulin, glucose transport and cell growth in CHO-V938D cells were as high as in insulin-stimulated control cells and no longer responsive to insulin stimulation. Phosphorylation of the beta subunit of the insulin receptor was also increased in CHO-V938D cells not exposed to insulin. These results support an essential role of the transmembrane domain of the insulin receptor in the transduction of insulin signaling.     

E-cadherin-mediated adhesion inhibits ligand-dependent activation of diverse receptor tyrosine kinases

E-cadherin is an essential adhesion protein as well as a tumor suppressor that is silenced in many cancers. Its adhesion-dependent regulation of signaling has not been elucidated. We report that E-cadherin can negatively regulate, in an adhesion-dependent manner, the ligand-dependent activation of divergent classes of receptor tyrosine kinases (RTKs), by inhibiting their ligand-dependent activation in association with decreases in receptor mobility and in ligand-binding affinity. E-cadherin did not regulate a constitutively active mutant RTK (Neu*) or the ligand-dependent activation of LPA receptors or muscarinic receptors, which are two classes of G protein-coupled receptors. EGFR regulation by E-cadherin was associated with complex formation between EGFR and E-cadherin that depended on the extracellular domain of E-cadherin but was independent of beta-catenin binding or p120-catenin binding. Transfection of E-cadherin conferred negative RTK regulation to human melanoma and breast cancer lines with downregulated endogenous E-cadherin. Abrogation of E-cadherin regulation may contribute to the frequent ligand-dependent activation of RTK in tumors.     

A single amino acid exchange inverts susceptibility of related receptor tyrosine kinases for the ATP site inhibitor STI-571

The tyrosine kinase inhibitor STI-571 potently blocks BCR-Abl, platelet-derived growth factor (PDGF) alpha- and beta-receptors, and c-Kit kinase activity. Flt3, a receptor tyrosine kinase closely related to PDGF receptors and c-Kit is, however, not inhibited by STI-571. Sequence alignments of different kinases and indications from the crystal structure of the STI-571 Abl kinase complex revealed amino acid residues that are probably crucial for this activity profile. It was predicted that Flt3 Phe-691 in the beta5 strand may sterically prevent interaction with STI-571. The point mutants Flt3 F691T and PDGFbeta-receptor T681F were constructed, and kinase assays showed that the Flt3 mutant but not the PDGFbeta-receptor mutant is inhibited by STI-571. Docking of STI-571 into computer models of the PDGFbeta-receptor and Flt3 kinase domains and comparison with the crystal structure of the STI-571 Abl kinase complex indicated very similar binding sites among the three nonphosphorylated kinases, suggesting corresponding courses of their Asp-Phe-Gly motifs and activation loops. Accordingly, we observed reduced sensitivity of preactivated compared with nonactivated PDGFR-beta for the inhibition by STI-571. Courses of the activation loop that collide with STI-571 binding explain its inactivity at other kinases as the insulin receptor. The binding site models of PDGFR-beta and Flt3 were applied to predict structural approaches for more selective PDGFbeta-receptor inhibitors.     

Signalling and functional diversity within the Axl subfamily of receptor tyrosine kinases

The related Axl, Sky and Mer receptor tyrosine kinases (RTKs) are increasingly being implicated in a host of discrete cellular responses including cell survival, proliferation, migration and phagocytosis. Furthermore, their ligands Gas6 and protein S are characteristically dependent on vitamin K for expression of their functions. The Gas6/Axl system is implicated in several types of human cancer as well as inflammatory, autoimmune, vascular and kidney diseases. Each member of the Axl RTK subfamily possesses distinct expression profiles as well as discrete functions. In this article, we review the knowledge so far on the intracellular signalling interactions and pathways concerning each of the Axl RTKs. In this way, we hope to gain a greater understanding of the mechanisms that set each of them apart, and that relay their associated functions.     

A single amino acid substitution in the beta-adrenergic receptor promotes partial agonist activity from antagonists

The family of G-protein-linked receptors includes many important pharmacological targets, of which the beta-adrenergic receptor is one of the best characterized. A better understanding of those factors that determine whether a ligand functions as an antagonist or as an agonist would facilitate the development of pharmaceutical agents that act at these receptors. Site-directed mutagenesis of the hamster beta 2-adrenergic receptor has implicated the conserved Asp113 residue in the third hydrophobic domain of the receptor in the interaction with cationic amine agonists and antagonists (Strader, C. D., Sigal, I. S., Candelore, M. R., Rands, E., Hill, W. S., and Dixon, R. A. F. (1988) J. Biol. Chem, 263, 10267-10271). We now report that substitution of Asp113 with a glutamic acid residue results in a mutant beta-adrenergic receptor which recognizes several known beta-adrenergic antagonists as partial agonists. This partial agonist activity requires the presence of a carboxylate side chain on the amino acid residue at position 113 and is not observed when an asparagine residue is substituted at this position. These observations support the existence of overlapping binding sites for agonists and antagonists on the beta-adrenergic receptor and demonstrate that genetic engineering of receptors can complement structure-activity studies of ligands in defining the molecular interactions involved in receptor activation.     

A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity

The receptor binding specificity of influenza viruses may be important for host restriction of human and avian viruses. Here, we show that the hemagglutinin (HA) of the virus that caused the 1918 influenza pandemic has strain-specific differences in its receptor binding specificity. The A/South Carolina/1/18 HA preferentially binds the alpha2,6 sialic acid (human) cellular receptor, whereas the A/New York/1/18 HA, which differs by only one amino acid, binds both the alpha2,6 and the alpha2,3 sialic acid (avian) cellular receptors. Compared to the conserved consensus sequence in the receptor binding site of avian HAs, only a single amino acid at position 190 was changed in the A/New York/1/18 HA. Mutation of this single amino acid back to the avian consensus resulted in a preference for the avian receptor.     

A single amino acid substitution in SecY stabilizes the interaction with SecA.

The SecYEG complex constitutes a protein conducting channel across the bacterial cytoplasmic membrane. It binds the peripheral ATPase SecA to form the translocase. When isoleucine 278 in transmembrane segment 7 of the SecY subunit was replaced by a unique cysteine, SecYEG supported an increased preprotein translocation and SecA translocation ATPase activity, and allowed translocation of a preprotein with a defective signal sequence. SecY(I278C)EG binds SecA with a higher affinity than normal SecYEG, in particular in the presence of ATP. The increased translocation activity of SecY(I278C)EG was confirmed in a purified system consisting of SecYEG proteoliposomes, while immunoprecipitation in detergent solution reveal that translocase-preprotein complexes are more stable with SecY(I278C) than with normal SecY. These data imply an important role for SecY transmembrane segment 7 in SecA binding. As improved SecA binding to SecY was also observed with the prlA4 suppressor mutation, it may be a general mechanism underlying signal sequence suppression.     

Five PDGF B amino acid substitutions convert PDGF A to a PDGF B-like transforming molecule.

We used site-directed mutagenesis to determine the minimum number of PDGF B residues needed to convert PDGF A to a potently transforming PDGF B-like molecule. Substitution of two PDGF B subdomains, 106-115 and 135-144, were found to be critical. These substitutions were sufficient to broaden the ability of PDGF A to activate beta as well as alpha platelet-derived growth factor (PDGF) receptors and increase its transforming efficiency to that of PDGF B. Within subdomain I, either PDGF B residues Arg-109 and Asn-115 or Arg-109, Leu-110, and Arg-113, in combination with subdomain II PDGF B residues Asn-136, Arg-137, and Arg-142 were identified as being essential. Those mutants with transforming ability comparable with PDGF B showed significantly lower efficiencies of beta receptor triggering. Thus, our studies identify a small number of PDGF B amino acids indispensable for beta PDGF receptor interaction and suggest that a low level of beta PDGF receptor activation is sufficient to dramatically increase PDGF transforming efficiency in NIH 3T3 cells.     

A single amino acid substitution reduces the superhelicity requirement of a replication initiator protein.

The origin of rolling circle replication in filamentous coliphage consists of a core origin that is absolutely required and an adjacent replication enhancer sequence that increases in vivo replication 30 to 100-fold. The core origin binds the initiator protein (gpII) which either nicks or relaxes negatively superhelical replicative form DNA (RFI). Nicking at the origin, but not relaxation, leads to initiation of DNA replication. Our results indicate that the ratio of nicking to relaxation (nicking-closing) in vitro depends on the superhelical density of the substrate. We have studied the effect of a single amino acid substitution in gpII, which allows wild-type levels of replication in the absence of the enhancer, on origin nicking and binding. The enhancer-independent mutation yields more nicking and less relaxation of RFI, compared to the wild-type protein. The mutant gpII also shows a reduced requirement for superhelicity of the substrate in the nicking reaction. At the same time, the mutant gpII increases the cooperativity of protein-protein interactions in origin binding. We propose that the relaxation activity of gpII negatively regulates replication initiation, and that both increase in the negative superhelicity of the substrate and action of the replication enhancer may antagonize the relaxation activity.     

A large compressibility change of protein induced by a single amino acid substitution.

The adiabatic compressibility (beta s) was determined, by means of the precise sound velocity and density measurements, for a series of single amino acid substituted mutant enzymes of Escherichia coli dihydrofolate reductase (DHFR) and aspartate aminotransferase (AspAT). Interestingly, the beta s values of both DHFR and AspAT were influenced markedly by the mutations at glycine-121 and valine-39, respectively, in which the magnitude of the change was proportional to the enzyme activity. This result demonstrates that the local change of the primary structure plays an important role in atomic packing and protein dynamics, which leads to the modified stability and enzymatic function. This is the first report on the compressibility of mutant proteins.     

A change in conformational dynamics underlies the activation of Eph receptor tyrosine kinases

Eph receptor tyrosine kinases (RTKs) mediate numerous developmental processes. Their activity is regulated by auto-phosphorylation on two tyrosines within the juxtamembrane segment (JMS) immediately N-terminal to the kinase domain (KD). Here, we probe the molecular details of Eph kinase activation through mutational analysis, X-ray crystallography and NMR spectroscopy on auto-inhibited and active EphB2 and EphA4 fragments. We show that a Tyr750Ala gain-of-function mutation in the KD and JMS phosphorylation independently induce disorder of the JMS and its dissociation from the KD. Our X-ray analyses demonstrate that this occurs without major conformational changes to the KD and with only partial ordering of the KD activation segment. However, conformational exchange for helix alphaC in the N-terminal KD lobe and for the activation segment, coupled with increased inter-lobe dynamics, is observed upon kinase activation in our NMR analyses. Overall, our results suggest that a change in inter-lobe dynamics and the sampling of catalytically competent conformations for helix alphaC and the activation segment rather than a transition to a static active conformation underlies Eph RTK activation.     

A single amino acid substitution in rhodopsin (lysine 248----leucine) prevents activation of transducin

In structure-function studies on bovine rhodopsin by in vitro site-specific mutagenesis, we have prepared three mutants in the cytoplasmic loop between the putative transmembrane helices E and F. In each mutant, charged amino acid residues were replaced by neutral residues: mutant 1, Glu239----Gln; mutant 2, Lys248----Leu; and mutant 3, Glu247----Gln, Lys248----Leu, and Glu249----Gln. The mutant rhodopsin genes were expressed in monkey kidney (COS-1) cells. After the addition of 11-cis-retinal to the cells, the rhodopsin mutants were purified by immunoaffinity adsorption. Each mutant gave a wild-type rhodopsin visible absorption spectrum. The mutants were assayed for their ability to stimulate the GTPase activity of transducin in a light-dependent manner. While mutants 1 and 3 showed wild-type activity, mutant 2 (Lys248----Leu) was inactive.     

Identification of a Frizzled-like cysteine rich domain in the extracellular region of developmental receptor tyrosine kinases.

In Drosophila, members of the Frizzled family of tissue-polarity genes encode proteins that appear to function as cell-surface receptors for Wnts. The Frizzled genes belong to the seven transmembrane class of receptors (7TMR) and have on their extracellular region a cysteine-rich domain that has been implicated as the Wnt binding domain. This region has a characteristic spacing of ten cysteines, which has also been identified in FrzB (a secreted antagonist of Wnt signaling) and Smoothened (another 7TMR, which is involved in suppression of the hedgehog pathway). We have identified, using BLAST, sequence similarity between the cysteine-rich domain of Frizzled and several receptor tyrosine kinases, which have roles in development. These include the muscle-specific receptor tyrosine kinase (MuSK), the neuronal specific kinase (NSK2), and ROR1 and ROR2. At present, the ligands for these developmental tyrosine kinases are unknown. Our results suggest that Wnt-like ligands may bind to these developmental tyrosine kinases.     

A single amino acid substitution in the activation loop defines the decoy characteristic of VEGFR-1/FLT-1

VEGFR-1 is a kinase-defective receptor tyrosine kinase (RTK) and negatively modulates angiogenesis by acting as a decoy receptor. The decoy characteristic of VEGFR-1 is required for normal development and angiogenesis. To date, there is no molecular explanation for this unusual characteristic of VEGFR-1. Here we show that the molecular mechanisms underlying the decoy characteristic of VEGFR-1 is linked to the replacement of a highly conserved amino acid residue in the activation loop. This amino acid is highly conserved among all the type III RTKs and corresponds to aspartic acid, but in VEGFR-1 it is substituted to asparagine. Mutation of asparagine (Asn(1050)) within the activation loop to aspartic acid promoted enhanced ligand-dependent tyrosine autophosphorylation and kinase activation in vivo and in vitro. The mutant VEGFR-1 (Asp(1050)) promoted endothelial cell proliferation but not tubulogenesis. It also displayed an oncogenic phenotype as its expression in fibroblast cells elicited transformation and colony growth. Furthermore, mutation of the invariable aspartic acid to asparagine in VEGFR-2 lowered the autophosphorylation of activation loop tyrosines 1052 and 1057. We propose that the conserved aspartic acid in the activation loop favors the transphosphorylation of the activation loop tyrosines, and its absence renders RTK to a less potent enzyme by disfavoring transphosphorylation of activation loop tyrosines.     

A limited spectrum of mutations causes constitutive activation of the yeast alpha-factor receptor

Activation of G protein coupled receptors (GPCRs) by binding of ligand is the initial event in diverse cellular signaling pathways. To examine the frequency and diversity of mutations that cause constitutive activation of one particular GPCR, the yeast alpha-factor receptor, we screened libraries of random mutations for constitutive alleles. In initial screens for mutant receptor alleles that exhibit signaling in the absence of added ligand, 14 different point mutations were isolated. All of these 14 mutants could be further activated by alpha-factor. Ten of the mutants also acquired the ability to signal in response to binding of desTrp(1)?Ala(3)?lpha-factor, a peptide that acts as an antagonist toward normal alpha-factor receptors. Of these 10 mutants, at least eight alleles residing in the third, fifth, sixth, and seventh transmembrane segments exhibit bona fide constitutive signaling. The remaining alleles are hypersensitive to alpha-factor rather than constitutive. They can be activated by low concentrations of endogenous alpha-factor present in MATa cells. The strongest constitutively active receptor alleles were recovered multiple times from the mutational libraries, and extensive mutagenesis of certain regions of the alpha-factor receptor did not lead to recovery of any additional constitutive alleles. Thus, only a limited number of mutations is capable of causing constitutive activation of this receptor. Constitutive and hypersensitive signaling by the mutant receptors is partially suppressed by coexpression of normal receptors, consistent with preferential association of the G protein with unactivated receptors.