Fc receptor gamma chain residues at the interface of the cytoplasmic and transmembrane domains affect association with FcalphaRI, surface expression, and function

The assembly of multiple subunit immunoreceptors is dependent on transmembrane interactions. The Fc receptor gamma (FcR-gamma) chain is a ubiquitous immune receptor tyrosine-based activation motif-containing dimeric subunit, gamma(2), which in humans associates with both the activating members of the leukocyte receptor cluster, including the IgA receptor FcalphaRI, and the classical Fc receptors, including the IgE receptor FcepsilonRI. This study identifies a new site in the transmembrane region of FcR-gamma that affects receptor assembly and surface expression with FcalphaRI but not with FcepsilonRI. The wild type complex, FcalphaRI-gamma(2)WT, remains robustly associated in both Brij-96 and Thesit detergent conditions. However, mutation of either Tyr(25) or Cys(26) of FcR-gamma, near the interface of the transmembrane and cytoplasmic regions, resulted in impaired FcR-gamma association with FcalphaRI. This association was disrupted in the presence of the detergent Brij-96 but was preserved in milder conditions using the detergent Thesit. Ligand-mediated cross-linking of the FcalphaRI-gamma(2)Y25F mutant receptor resulted in diminished signal transduction, including an abnormal calcium response, compared with the FcalphaRI-gamma(2)WT receptor. Furthermore, the FcalphaRI-gamma(2)Y25F mutant receptor was expressed at the cell surface at approximately 10% of that of the wild type, whereas the surface expression of FcepsilonRI-gamma(2)Y25F was not significantly different from the wild type. In contrast, although the FcalphaRI-gamma(2)C26S mutant was also less stably associated, it was not reduced in surface expression or function. Thus, these TM residues of FcR-gamma are important for association with FcalphaRI and probably other activating LRC members but not with the classical FcR, FcepsilonRI.     

Crystal structure of the ectodomain of human FcalphaRI

Human FcalphaRI (CD89) is the receptor specific for IgA, an immunoglobulin that is abundant in mucosa and is also found in high concentrations in serum. Although FcalphaRI is an immunoglobulin Fc receptor (FcR), it differs in many ways from FcRs for other immunoglobulin classes. The genes of most FcRs are located on chromosome 1 at 1q21-23, whereas FcalphaRI is on chromosome 19, at 19q13.4, a region called the leukocyte receptor complex, because it is clustered with several leukocyte receptor families including killer cell inhibitory receptors (KIRs) and leukocyte Ig-like receptors (LIRs). The amino acid sequence of FcalphaRI shares only 20% homology with other FcRs but it has around 35% homology with its neighboring LIRs and KIRs. In this work, we analyzed the crystal structure of the ectodomain of FcalphaRI and examined structure similarities between FcalphaRI and KIR2DL1, KIR2DL2 and LIR-1. Our data show that FcalphaRI, KIRs, and LIRs share a common hydrophobic core in their interdomain interface, and FcalphaRI is evolutionally closer to LIR than KIR.     

A competitive mechanism for staphylococcal toxin SSL7 inhibiting the leukocyte IgA receptor, Fc alphaRI, is revealed by SSL7 binding at the C alpha2/C alpha3 interface of IgA

Leukocyte recruitment and effector functions like phagocytosis and respiratory burst are key elements of immunity to infection. Pathogen survival is dependent upon the ability to overwhelm, evade or inhibit the immune system. Pathogenic group A and group B streptococci are well known to produce virulence factors that block the binding of IgA to the leukocyte IgA receptor, Fc alphaRI, thereby inhibiting IgA-mediated immunity. Recently we found Staphylococcus aureus also interferes with IgA-mediated effector functions as the putative virulence factor SSL7 also binds IgA and blocks binding to Fc alphaRI. Herein we report that SSL7 and Fc alphaRI bind many of the same key residues in the Fc region of human IgA. Residues Leu-257 and Leu-258 in domain C alpha2 and residues 440-443 PLAF in C alpha3 of IgA lie at the C alpha2/C alpha3 interface and make major contributions to the binding of both the leukocyte receptor Fc alphaRI and SSL7. It is remarkable this S. aureus IgA binding factor and unrelated factors from streptococci are functionally convergent, all targeting a number of the same residues in the IgA Fc, which comprise the binding site for the leukocyte IgA receptor, Fc alphaRI.     

The interaction of Fc alpha RI with IgA and its implications for ligand binding by immunoreceptors of the leukocyte receptor cluster

This study defines the molecular basis of the FcalphaRI (CD89):IgA interaction, which is distinct from that of the other leukocyte Fc receptors and their Ig ligands. A comprehensive analysis using both cell-free (biosensor) and cell-based assays was used to define and characterize the IgA binding region of FcalphaRI. Biosensor analysis of mutant FcalphaRI proteins showed that residues Y35, Y81, and R82 were essential for IgA binding, and R52 also contributed. The role of the essential residues (Y35 and R82) was confirmed by analysis of mutant receptors expressed on the surface of mammalian cells. These receptors failed to bind IgA, but were detected by the mAb MY43, which blocks IgA binding to FcalphaRI, indicating that its epitope does not coincide with these IgA binding residues. A homology model of the ectodomains of FcalphaRI was generated based on the structures of killer Ig-like receptors, which share 30-34% identity with FcalphaRI. Key structural features of killer Ig-like receptors are appropriately reproduced in the model, including the structural conservation of the interdomain linker and hydrophobic core (residues V17, V97, and W183). In this FcalphaRI model the residues forming the IgA binding site identified by mutagenesis form a single face near the N-terminus of the receptor, distinct from other leukocyte Fc receptors where ligand binding is in the second domain. This taken together with major differences in kinetics and affinity for IgA:FcalphaRI interaction that were observed depending on whether FcalphaRI was immobilized or in solution suggest a mode of interaction unique among the leukocyte receptors.     

The structure of a human type III Fcgamma receptor in complex with Fc

Fcgamma receptors mediate antibody-dependent inflammatory responses and cytotoxicity as well as certain autoimmune dysfunctions. Here we report the crystal structure of a human Fc receptor (FcgammaRIIIB) in complex with an Fc fragment of human IgG1 determined from orthorhombic and hexagonal crystal forms at 3.0- and 3.5-A resolution, respectively. The refined structures from the two crystal forms are nearly identical with no significant discrepancies between the coordinates. Regions of the C-terminal domain of FcgammaRIII, including the BC, C'E, FG loops, and the C' beta-strand, bind asymmetrically to the lower hinge region, residues Leu(234)-Pro(238), of both Fc chains creating a 1:1 receptor-ligand stoichiometry. Minor conformational changes are observed in both the receptor and Fc upon complex formation. Hydrophobic residues, hydrogen bonds, and salt bridges are distributed throughout the receptor.Fc interface. Sequence comparisons of the receptor-ligand interface residues suggest a conserved binding mode common to all members of immunoglobulin-like Fc receptors. Structural comparison between FcgammaRIII.Fc and FcepsilonRI.Fc complexes highlights the differences in ligand recognition between the high and low affinity receptors. Although not in direct contact with the receptor, the carbohydrate attached to the conserved glycosylation residue Asn(297) on Fc may stabilize the conformation of the receptor-binding epitope on Fc. An antibody-FcgammaRIII model suggests two possible ligand-induced receptor aggregations.     

Pairwise interactions between neuronal alpha(7) acetylcholine receptors and alpha-conotoxin PnIB

This work uses alpha-conotoxin PnIB to probe the agonist binding site of neuronal alpha(7) acetylcholine receptors. We mutated the 13 non-cysteine residues in CTx PnIB, expressed alpha(7)/5-hydroxytryptamine-3 homomeric receptors in 293 HEK cells, and measured binding of each mutant toxin to the expressed receptors by competition against the initial rate of (125)I-alpha-bungarotoxin binding. The results reveal that residues Ser-4, Leu-5, Pro-6, Pro-7, Ala-9, and Leu-10 endow CTx PnIB with affinity for alpha(7)/5-hydroxytryptamine-3 receptors; side chains of these residues cluster in a localized region within the three-dimensional structure of CTx PnIB. We next mutated key residues in the seven loops of alpha(7) that converge at subunit interfaces to form the agonist binding site. The results reveal predominant contributions by residues Trp-149 and Tyr-93 in alpha(7) and smaller contributions by Ser-34, Arg-186, Tyr-188, and Tyr-195. To identify pairwise interactions that stabilize the receptor-conotoxin complex, we measured binding of receptor and toxin mutations and analyzed the results by double mutant cycles. The results reveal a single dominant interaction between Leu-10 of CTx PnIB and Trp-149 of alpha(7) that anchors the toxin to the binding site. We also find weaker interactions between Pro-6 of CTx PnIB and Trp-149 and between both Pro-6 and Pro-7 and Tyr-93 of alpha(7). The overall results demonstrate that a localized hydrophobic region in CTx PnIB interacts with conserved aromatic residues on one of the two faces of the alpha(7) binding site.     

Interacting helical surfaces of the transmembrane segments of subunits a and c' of the yeast V-ATPase defined by disulfide-mediated cross-linking

Proton translocation by the vacuolar (H+)-ATPase (or V-ATPase) has been shown by mutagenesis to be dependent upon charged residues present within transmembrane segments of subunit a as well as the three proteolipid subunits (c, c', and c"). Interaction between R735 in TM7 of subunit a and the glutamic acid residue in the middle of TM4 of subunits c and c' or TM2 of subunit c" has been proposed to be essential for proton release to the luminal compartment. In order to determine whether the helical face of TM7 of subunit a containing R735 is capable of interacting with the helical face of TM4 of subunit c' containing the essential glutamic acid residue (Glu-145), cysteine-mediated cross-linking between these subunits in yeast has been performed. Cys-less forms of subunits a and c' as well as forms containing unique cysteine residues were constructed, introduced together into a strain disrupted in both endogenous subunits, and tested for growth at neutral pH, for assembly competence and for cross-linking in the presence of cupric-phenanthroline by SDS-PAGE and Western blot analysis. Four different cysteine mutants of subunit a were each tested pairwise with ten different unique cysteine mutants of subunit c'. Strong cross-linking was observed for the pairs aS728C/c'I142C, aA731C/c'E145C, aA738C/c'F143C, aA738C/c'L147C, and aL739C/c'L147C. Partial cross-linking was observed for an additional 13 of 40 pairs analyzed. When arrayed on a helical wheel diagram, the results suggest that the helical face of TM7 of subunit a containing Arg-735 interacts with the helical face of TM4 of subunit c' centered on Val-146 and bounded by Glu-145 and Leu-147. The results are consistent with a possible rotational flexibility of one or both of these transmembrane segments as well as some flexibility of movement perpendicular to the membrane.     

Molecular dissection of the FcRbeta signaling amplifier

Human high affinity IgE receptors are expressed as two different isoforms: the tetrameric isoform, alphabetagamma(2), or the trimeric isoform, alphagamma(2). The alpha chain is the IgE binding subunit, whereas the FcRbeta and FcRgamma chains are the signaling modules. Both FcRbeta and FcRgamma contain immunoreceptor tyrosine-based activation motifs (ITAM), but the beta ITAM differs from canonical ITAMs in two ways; the spacing between the two canonical tyrosines harbors a third tyrosine, and it is one amino acid shorter than in canonical ITAMs, making it unfit to bind the tandem SH2 of Syk. We have shown that FcRbeta functions as an amplifier of the FcRgamma signaling function. However, the molecular mechanism of this amplification remains unclear. Here we show that mutation of the three tyrosines (Tyr-219, Tyr-225, and Tyr-229) in the beta ITAM essentially converts alphabetagamma(2)into an alphagamma(2) complex in terms of Lyn recruitment, FcRgamma phosphorylation, Syk activation, and calcium mobilization. Tyr-219 is the most critical residue in this regard. In addition, a detailed analysis of the dynamics of calcium mobilization suggests a possible inhibitory role for Tyr-225, which becomes apparent when Tyr-219 is mutated. Thus, the signaling amplification function of FcRbeta is mainly encoded in Tyr-219 and in its capacity to recruit Lyn. In turn, this Tyr-219-mediated Lyn recruitment enhances gamma chain phosphorylation, Syk activation, and calcium mobilization. The two other tyrosines appear to have a modulating function that remains to be fully assessed.     

Lipid rafts orchestrate signaling by the platelet receptor glycoprotein VI

The platelet collagen receptor glycoprotein VI (GPVI) couples to the immune receptor adaptor Fc receptor gamma-chain (FcRgamma) and signals using many of the same intracellular signaling molecules as immune receptors. Studies of immune receptor signaling have revealed a critical role for specialized areas of the cell membrane known as lipid rafts, which are enriched in essential signaling molecules. However, the role of lipid rafts in signaling in nonimmune cells such as platelets remains poorly defined. This study shows that GPVI-FcRgamma does not constitutively associate with rafts, but is recruited to lipid rafts following receptor stimulation in both GPVI-expressing RBL-2H3 cells and human platelets. FcRgamma is required for GPVI association with lipid rafts, as mutant GPVI receptors that do not couple to FcRgamma were unable to associate with lipid rafts after receptor clustering. Following GPVI stimulation in platelets, virtually all phosphorylated FcRgamma was found in lipid rafts, but inhibition of FcRgamma phosphorylation did not block receptor association with lipid rafts. This work demonstrates that lipid rafts orchestrate GPVI receptor signaling in platelets in a manner analogous to immune cell receptors and supports a model of GPVI signaling in which FcRgamma phosphorylation is controlled by ligand-dependent association with lipid rafts.     

Molecular Characterization of Oxysterol Binding to the Epstein-Barr Virus-induced Gene 2 (GPR183)

Oxysterols are oxygenated cholesterol derivates that are emerging as a physiologically important group of molecules. Although they regulate a range of cellular processes, only few oxysterol-binding effector proteins have been identified, and the knowledge of their binding mode is limited. Recently, the family of G protein-coupled seven transmembrane-spanning receptors (7TM receptors) was added to this group. Specifically, the Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) was shown to be activated by several oxysterols, most potently by 7α,25-dihydroxycholesterol (7α,25-OHC). Nothing is known about the binding mode, however. Using mutational analysis, we identify here four key residues for 7α,25-OHC binding: Arg-87 in TM-II (position II:20/2.60), Tyr-112 and Tyr-116 (positions III:09/3.33 and III:13/3.37) in TM-III, and Tyr-260 in TM-VI (position VI:16/6.51). Substituting these residues with Ala and/or Phe results in a severe decrease in agonist binding and receptor activation. Docking simulations suggest that Tyr-116 interacts with the 3β-OH group in the agonist, Tyr-260 with the 7α-OH group, and Arg-87, either directly or indirectly, with the 25-OH group, although nearby residues likely also contribute. In addition, Tyr-112 is involved in 7α,25-OHC binding but via hydrophobic interactions. Finally, we show that II:20/2.60 constitutes an important residue for ligand binding in receptors carrying a positively charged residue at this position. This group is dominated by lipid- and nucleotide-activated receptors, here exemplified by the CysLTs, P2Y12, and P2Y14. In conclusion, we present the first molecular characterization of oxysterol binding to a 7TM receptor and identify position II:20/2.60 as a generally important residue for ligand binding in certain 7TM receptors.     

Regulation of the oncogenic activity of the cellular src protein requires the correct spacing between the kinase domain and the C-terminal phosphorylated tyrosine (Tyr-527).

Repression of the tyrosine kinase activity of the cellular src protein (pp60c-src) depends on the phosphorylation of a tyrosine residue (Tyr-527) near the carboxy terminus. Tyr-527 is located 11 residues C terminal from the genetically defined end of the kinase domain (Leu-516) and is therefore in a negative regulatory region. Because the precise sequence of amino acids surrounding Tyr-527 appears to be unimportant for regulation, we hypothesized that the conformational constraints induced by phosphorylated Tyr-527 may require the correct spacing between the kinase domain (Leu-516) and Tyr-527. In this report, we show that deletions at residue 518 of two, four, or seven amino acids or insertions at this residue of two or four amino acids activated the kinase activity and thus the transforming potential of pp60c-src. As is the case for the prototype transforming variant, pp60527F, activation caused by these deletions or insertions was abolished when Tyr-416 (the autophosphorylation site) was changed to phenylalanine. In comparison with wild-type pp60c-src, the src proteins containing the alterations at residue 518 showed a lower phosphorylation state at Tyr-527 regardless of whether residue 416 was a tyrosine or a phenylalanine. Mechanisms dealing with the importance of spacing between the kinase domain and Tyr-527 are discussed.     

The neurokinin-1 and neurokinin-2 receptor binding sites of MDL103,392 differ

Several small molecule non-peptide antagonists of the NK-1 and NK-2 receptors have been developed. Mutational analysis of the receptor protein sequence has led to the conclusion that the binding site for these non-peptide antagonists lies within the bundle created by transmembrane domains IV–VII of the receptor and differs from the binding sites of peptide agonists and antagonists. The current investigation uses site-directed mutagenesis of the NK-1 and NK-2 receptors to elucidate the amino acids that are important for binding and functional activity of the first potent dual NK-1/NK-2 antagonist MDL103,392. The amino acids found to be important for MDL103,392 binding to the NK-1 receptor are Gln-165, His-197, Leu-203, Ile-204, Phe-264, His-265 and Tyr-272. The amino acids found to be important for MDL103,392 binding to the NK-2 receptor are Gln-166, His-198, Tyr-266 and Tyr-289. While residues in transmembrane (TM) domains IV and V are important in both receptors (Gln-165/166 and His-197/198), residues in TM V and VI are more important for the NK-1 receptor and residues in TM VII play a more important role in the NK-2 receptor. These data are the first report of the analysis of the binding site of a dual tachykinin receptor antagonist and indicate that a single compound (MDL103,392) binds to each receptor in a different manner despite there being a high degree of homology in the transmembrane bundles. In addition, this is the first report in which a model for the binding of a non-peptide antagonist to the NK-2 receptor is proposed.     

Maltose-binding protein interacts simultaneously and asymmetrically with both subunits of the Tar chemoreceptor

The Tar chemotactic signal transducer of Escherichia coli mediates attractant responses to L-aspartate and to maltose. Aspartate binds across the subunit interface of the periplasmic receptor domain of a Tar homodimer. Maltose, in contrast, first binds to the periplasmic maltose-binding protein (MBP), which in its ligand-stabilized closed form then interacts with Tar. Intragenic complementation was used to determine the MBP-binding site on the Tar dimer. Mutations causing certain substitutions at residues Tyr-143, Asn-145, Gly-147, Tyr-149, and Phe-150 of Tar lead to severe defects in maltose chemotaxis, as do certain mutations affecting residues Arg-73, Met-76, Asp-77, and Ser-83. These two sets of mutations defined two complementation groups when the defective proteins were co-expressed at equal levels from compatible plasmids. We conclude that MBP contacts both subunits of the Tar dimer simultaneously and asymmetrically. Mutations affecting Met-75 could not be complemented, suggesting that this residue is important for association of MBP with each subunit of the Tar dimer. When the residues involved in interaction with MBP were mapped onto the crystal structure of the Tar periplasmic domain, they localized to a groove at the membrane-distal apex of the domain and also extended onto one shoulder of the apical region.     

An activation switch in the rhodopsin family of G protein-coupled receptors: the thyrotropin receptor

We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of thyrotropin receptor (TSHr) activation. Asn-7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche+ conformation is maintained by interactions with residues Thr-6.43 and Asp-6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of approximately 14 and approximately 10 relative to wild type TSHr, respectively. Upon receptor activation Asn-7.49 adopts the trans conformation to interact with Asp-2.50 and a putatively charged residue that remains to be identified. In addition, the conserved Leu-2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state because the L2.46A mutation increases constitutive activity by a factor of approximately 13 relative to wild type TSHr. As residues Leu-2.46, Asp-2.50, and Asn-7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.     

Structural evidence that colicin A protein binds to a novel binding site of TolA protein in Escherichia coli periplasm

The Tol assembly of proteins is an interacting network of proteins located in the Escherichia coli cell envelope that transduces energy and contributes to cell integrity. TolA is central to this network linking the inner and outer membranes by interactions with TolQ, TolR, TolB, and Pal. Group A colicins, such as ColA, parasitize the Tol network through interactions with TolA and/or TolB to facilitate translocation through the cell envelope to reach their cytotoxic site of action. We have determined the first structure of the C-terminal domain of TolA (TolAIII) bound to an N-terminal ColA polypeptide (TA(53-107)). The interface region of the TA(53-107)-TolAIII complex consists of polar contacts linking residues Arg-92 to Arg-96 of ColA with residues Leu-375-Pro-380 of TolA, which constitutes a β-strand addition commonly seen in more promiscuous protein-protein contacts. The interface region also includes three cation-π interactions (Tyr-58-Lys-368, Tyr-90-Lys-379, Phe-94-Lys-396), which have not been observed in any other colicin-Tol protein complex. Mutagenesis of the interface residues of ColA or TolA revealed that the effect on the interaction was cumulative; single mutations of either partner had no effect on ColA activity, whereas mutations of three or more residues significantly reduced ColA activity. Mutagenesis of the aromatic ring component of the cation-π interacting residues showed Tyr-58 of ColA to be essential for the stability of complex formation. TA(53-107) binds on the opposite side of TolAIII to that used by g3p, ColN, or TolB, illustrating the flexible nature of TolA as a periplasmic hub protein.     

Radioiodination of tyrosine residue(s) of ox testis and of wheat germ calmodulins

Radioiodination of the two tyrosine residues (Tyr-99 and Tyr-138) of ox testis calmodulin was performed using several methods, and studied through the specific activity, and the [125I]iodoamino acid analysis of the radiolabeled calmodulins. Hydrolysis by thrombin of 125I-calmodulin labeled by the lactoperoxidase method and subsequent isolation of peptides TM1 and TM2 by gel electrophoresis showed preferential labeling by 125I of Tyr-99 (TM1) over Tyr-138 (TM2). Analysis of [125I]iodoamino acids of radiolabeled TM1, TM2 and calmodulin demonstrated that [125I]monoiodotyrosine was predominant, the remainder being [125I]diiodotyrosine. Radioiodination of wheat germ calmodulin, which contains a single tyrosine residue (Tyr-139), showed that only TM2 was labeled by 125I on the Tyr-139 residue and also on the His-108 residue (radiolabeled monoiodotyrosine, diiodotyrosine and monoiodohistidine being present).     

Tyrosine phosphorylation inhibits the interaction of 14-3-3 proteins with the plant plasma membrane H+-ATPase

Interaction of 14-3-3 proteins with their targets depends not only on the phosphorylation status of the target but also on that of 14-3-3 (Fu et al., 2000). In this work we demonstrated that the maize 14-3-3 isoform GF14-6 is a substrate of the tyrosine kinase insulin growth factor receptor 1. By means of site-directed mutants of GF14-6, we identified Tyr-137 as the specific tyrosine residue phosphorylated by the insulin growth factor receptor 1. Phosphorylation of GF14-6 on Tyr-137 lowered its affinity for a peptide mimicking the 14-3-3 binding site of the plant plasma membrane H+-ATPase. Moreover, phosphorylation in planta of 14-3-3 tyrosine residues, resulting from incubation with the tyrosine phosphatase inhibitor, phenylarsine oxide, decreased their association to the H+-ATPase.     

Amino acid replacements in yeast iso-1-cytochrome c. Comparison with the phylogenetic series and the tertiary structure of related cytochromes c

The structural and folding requirements of eukaryotic cytochromes c have been investigated by determining the appropriate DNA sequences of a collection of 46 independent cyc 1 missense mutations obtained in the yeast Saccharomyces cerevisiae and by deducing the corresponding amino acid replacements that abolish function of iso-1-cytochrome c. A total of 33 different replacements at 19 amino acid positions were uncovered in this and previous studies. Because all of these nonfunctional iso-1-cytochromes c are produced at far below the normal level and because a representative number are labile in vitro, most of the replacements appear to be affecting stability of the protein or heme attachment. By considering the tertiary structure of related cytochromes c, the loss of function of most of the mutant iso-1-cytochromes c could be attributed to either replacements of critical residues that directly interact with the heme group or to replacements that disrupt the proper folding of the protein. The replacements of residues interacting with the heme group include those required for covalent attachment (Cys-19 and Cys-22), ligand formation (His-23 and Met-85), and formation of the immediate heme environment (Leu-37, Tyr-53, Trp-64, and Leu-73). Proper folding of the protein is prevented by replacements of glycine residues at sites that cannot accommodate side chains (Gly-11 and Gly-34); by replacements of residues with proline, which limit the torsion angle (Leu-14 and His-38); and by replacements apparently unable to direct the local folding of the backbone into the proper conformation (Pro-35, Tyr-72, Asn-75, Pro-76, Lys-84, Leu-99, and Leu-103). Even though most of the missense mutations occurred at sites corresponding to evolutionarily invariant or conserved residues, a consideration of the replacements in functional revertants indicates that the requirement for residues evolutionarily preserved is less stringent than commonly assumed.     

Application of peptide-mediated ring current shifts to the study of neurophysin-peptide interactions: a partial model of the neurophysin-peptide complex

Perdeuteriated peptides were synthesized that are capable of binding to the hormone binding site of neurophysin but that differ in the position of aromatic residues. The binding of these peptides to bovine neurophysin I and its des-1-8 derivative was studied by proton nuclear magnetic resonance spectroscopy in order to identify protein residues near the binding site through the observation of differential ring current effects on assignable protein resonances. Phenylalanine in position 3 of bound peptides was shown to induce significant ring current shifts in several resonances assignable to the 1-8 sequence, including those of Leu-3 and/or Leu-5, but was without effect on Tyr-49 ring protons. The magnitude of these shifts was dependent on the identity of peptide residue 1. By contrast, the sole demonstrable direct effect of an aromatic residue in position 1 was a downfield shift in Tyr-49 ring protons. Study of peptide binding to des-1-8-neurophysin demonstrated similar conformations of native and des-1-8 complexes except for the environment of Tyr-49, confirmed the peptide-induced ring current shift assignments in native neurophysin, and indicated an effect of binding on Thr-9. These observations are integrated with other results to provide a partial model of neurophysin-peptide complexes that places the ring of Tyr-49 at a distance 5-10 A from residue 1 of bound peptide and that places both the 1-8 sequence and the protein backbone region containing Tyr-49 proximal to each other and to peptide residue 3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tyrosine phosphorylation of the beta2 subunit of clathrin adaptor complex AP-2 reveals the role of a di-leucine motif in the epidermal growth factor receptor trafficking

Tyrosine phosphorylation of the beta2 subunit of clathrin adaptor complex AP-2 was detected in three types of cells treated with epidermal growth factor (EGF). The tyrosine phosphorylation was observed during recruitment of EGF receptors into coated pits at 4 degrees C and reached maximum at 37 degrees C at post-recruitment stages of endocytosis. An inhibitor of EGF receptor kinase completely abolished this phosphorylation in all cell types, whereas the inhibitor of Src family kinases partially inhibited beta2 phosphorylation in A-431 cells but not in HeLa cells. By using beta2 subunit tagged with yellow fluorescent protein that is effectively assembled into AP-2 complex, the major phosphorylation site of beta2 was mapped to Tyr-6. Analysis of cells expressing dominant-interfering mutant mu2 subunit of AP-2 suggested that beta2 phosphorylation is partially mediated by the receptor interaction with the mu2 subunit. Mutation of leucine residues 1010 and 1011 motif in the EGF receptor resulted in the severe inhibition of beta2 tyrosine phosphorylation. From these data, we propose that interactions of the EGF receptor with AP-2 mediated by the receptor 974YRAL and di-leucine motifs may contribute to beta2 tyrosine phosphorylation. Surprisingly, mutation of the Leu-1010/Leu-1011 motif resulted in impaired degradation of EGF receptors, suggesting the role of this motif in lysosomal targeting of the receptor.