Association of INAD with NORPA is essential for controlled activation and deactivation of Drosophila phototransduction in vivo

Visual transduction in Drosophila is a G protein-coupled phospholipase C-mediated process that leads to depolarization via activation of the transient receptor potential (TRP) calcium channel. Inactivation-no-afterpotential D (INAD) is an adaptor protein containing PDZ domains known to interact with TRP. Immunoprecipitation studies indicate that INAD also binds to eye-specific protein kinase C and the phospholipase C, no-receptor-potential A (NORPA). By overlay assay and site-directed mutagenesis we have defined the essential elements of the NORPA–INAD association and identified three critical residues in the C-terminal tail of NORPA that are required for the interaction. These residues, Phe-Cys-Ala, constitute a novel binding motif distinct from the sequences recognized by the PDZ domain in INAD. To evaluate the functional significance of the INAD–NORPA association in vivo, we generated transgenic flies expressing a modified NORPA, NORPAC1094S, that lacks the INAD interaction. The transgenic animals display a unique electroretinogram phenotype characterized by slow activation and prolonged deactivation. Double mutant analysis suggests a possible inaccessibility of eye-specific protein kinase C to NORPAC1094S, undermining the observed defective deactivation, and that delayed activation may similarly result from NORPAC1094S being unable to localize in close proximity to the TRP channel. We conclude that INAD acts as a scaffold protein that facilitates NORPA–TRP interactions required for gating of the TRP channel in photoreceptor cells.

Phosphorylation of Tyrosine 992, 1068, and 1086 Is Required for Conformational Change of the Human Epidermal Growth Factor Receptor C-Terminal Tail

We reported previously that a conformation-specific antibody, Ab P2, to a 16-amino acid peptide (Glu-Gly-Tyr-Lys-Lys-Lys-Tyr-Gln-Gln-Val-Asp-Glu-Glu-Phe-Leu-Arg) of the cytoplasmic domain of the β-type platelet-derived growth factor receptor also recognizes the epidermal growth factor (EGF) receptor. Although the antibody is not directed to phosphotyrosine, it recognizes in immunoprecipitation the activated and hence phosphorylated form of both receptors. In P2 peptide, there are two tripeptide sequences, Asp-Glu-Glu and Tyr-Gln-Gln, that are also present in the EGF receptor. Our present studies using either EGF receptor C-terminal deletion mutants or point mutations (Tyr→Phe) and our previous studies on antibody inhibition by P2-derived peptides suggest that Gln-Gln in combination with Asp-Glu-Glu forms a high-affinity complex with Ab P2 and that such complex formation is dependent on tyrosine phosphorylation. Of the five phosphate acceptor sites in the EGF receptor, clustered in the extreme C-terminal tail, phosphorylation of three tyrosine residues (992, 1068, and 1086) located between Asp-Glu-Glu and Gln-Gln is necessary for Ab P2 binding. In contrast, the acceptor sites Tyr 1173 and 1148 play no role in the conformation change. Asp-Glu-Glu and Gln-Gln are located 169 amino acids apart, and it is highly likely that the interactions among three negatively charged phosphotyrosine residues in the receptor C terminus may result in the bending of the peptide chain in such a way that these two peptides come close to each other to form an antibody-binding site. Such a possibility is also supported by our finding that receptor dephosphorylation results in complete loss of Ab P2–binding activity. In conclusion, we have identified a domain within the cytoplasmic part of the EGF receptor whose conformation is altered by receptor phosphorylation; furthermore, we have identified the tyrosine residues that positively regulate this conformation.

Recognition Signal for C-Mannosylation of Trp-7 in RNase 2 Consists of Sequence Trp-x-x-Trp

C2-α-Mannosyltryptophan was discovered in human RNase 2, an enzyme that occurs in eosinophils and is involved in host defense. It represents a novel way of attaching carbohydrate to a protein in addition to the well-known N- and O-glycosylations. The reaction is specific, as in RNase 2 Trp-7, but never Trp-10, which is modified. In this article, we address which structural features provide the specificity of the reaction. Expression of chimeras of RNase 2 and nonglycosylated RNase 4 and deletion mutants in HEK293 cells identified residues 1–13 to be sufficient for C-mannosylation. Site-directed mutagenesis revealed the sequence Trp-x-x-Trp, in which the first Trp becomes mannosylated, as the specificity determinant. The Trp residue at position +3 can be replaced by Phe, which reduces the efficiency of the reaction threefold. Interpretation of the data in the context of the three-dimensional structure of RNase 2 strongly suggests that the primary, rather than the tertiary, structure forms the determinant. The sequence motif occurs in 336 mammalian proteins currently present in protein databases. Two of these proteins were analyzed protein chemically, which showed partial C-glycosylation of recombinant human interleukin 12. The frequent occurrence of the protein recognition motif suggests that C-glycosides could be part of the structure of more proteins than assumed so far.

Highly Stoichiometric, Stable, and Specific Association of Integrin α3β1 with CD151 Provides a Major Link to Phosphatidylinositol 4-Kinase, and May Regulate Cell Migration

Here we describe an association between α3β1 integrin and transmembrane-4 superfamily (TM4SF) protein CD151. This association is maintained in relatively stringent detergents and thus is remarkably stable in comparison with previously reported integrin–TM4SF protein associations. Also, the association is highly specific (i.e., observed in vitro in absence of any other cell surface proteins), and highly stoichiometric (nearly 90% of α3β1 associated with CD151). In addition, α3β1 and CD151 appeared in parallel on many cell lines and showed nearly identical skin staining patterns. Compared with other integrins, α3β1 exhibited a considerably higher level of associated phosphatidylinositol-4-kinase (PtdIns 4-kinase) activity, most of which was removed upon immunodepletion of CD151. Specificity for CD151 and PtdIns 4-kinase association resided in the extracellular domain of α3β1, thus establishing a novel paradigm for the specific recruitment of an intracellular signaling molecule. Finally, antibodies to either CD151 or α3β1 caused a ∼88–92% reduction in neutrophil motility in response to f-Met-Leu-Phe on fibronectin, suggesting an functionally important role of these complexes in cell migration.

Identification of surface residues mediating tissue factor binding and catalytic function of the serine protease factor VIIa

Factor VIIa (VIIa), the serine protease that initiates the coagulation pathways, is catalytically activated upon binding to its cell surface receptor and cofactor tissue factor (TF). This study provides a comprehensive analysis of the functional surface of VIIa by alanine scanning mutagenesis of 112 residues. Residue side chains were defined which contribute to TF binding and factor X hydrolysis. Energetically important binding contacts at the interface with TF were identified in the first epidermal growth factor domain of VIIa (Gln-64, Ile-69, Phe-71, Arg-79) and in the protease domain (Arg-277, Met-306, Asp-309). The observed energetic defects are in good agreement with the corresponding residues in TF, suggesting that the VIIa light chain plays a prominent role in high affinity binding of cofactor. Mutation of protease domain interface residues indicated that TF allosterically influences the active site of VIIa. Stabilization of a labile zymogen to enzyme transition could explain the activating effect of TF on VIIa catalytic function. Residues important for factor X hydrolysis were found in three regions of the protease domain: (i) specificity determinants in the catalytic cleft and adjacent loops, (ii) an exosite near the TF binding site, and (iii) a large electronegative exosite which is in a position analogous to the basic exosite I of thrombin. TF regions involved in factor X activation are positioned on the same face of the TF·VIIa complex as the two exosites identified on the protease domain surface, providing evidence for an extended interaction of TF·VIIa with macromolecular substrate.

A general method for determining helix packing in membrane proteins in situ: Helices I and II are close to helix VII in the lactose permease of Escherichia coli

It was previously shown that coexpression of the lactose permease of Escherichia coli in two contiguous fragments leads to functional complementation. We demonstrate here that site-directed thiol crosslinking of coexpressed permease fragments can be used to determine helix proximity in situ without the necessity of purifying the permease. After coexpression of the six N-terminal (N6) and six C-terminal (C6) transmembrane helices, each with a single Cys residue, crosslinking was carried out in native membranes and assessed by the mobility of anti-C-terminal-reactive polypeptides on immunoblots. A Cys residue at position 242 or 245 (helix VII) forms a disulfide with a Cys residue at either position 28 or 29 (helix I), but not with a Cys residue at position 27, which is on the opposite face of helix I, thereby indicating that the face of helix I containing Pro-28 and Phe-29 is close to helix VII. Similarly, a Cys residue at position 242 or 245 (helix VII) forms a disulfide with a Cys residue at either position 52 or 53 (helix II), but not with a Cys residue at position 54. Furthermore, low-efficiency crosslinking is observed between a Cys residue at position 52 or 53 and a Cys residue at position 361 (helix XI). The results indicate that helix VII lies in close proximity to both helices I and II and that helix II is also close to helix XI. The method should be applicable to a number of different polytopic membrane proteins.

Unexpected crucial role of residue 225 in serine proteases

Residue 225 in serine proteases of the chymotrypsin family is Pro or Tyr in more than 95% of nearly 300 available sequences. Proteases with Y225 (like some blood coagulation and complement factors) are almost exclusively found in vertebrates, whereas proteases with P225 (like degradative enzymes) are present from bacteria to human. Saturation mutagenesis of Y225 in thrombin shows that residue 225 affects ligand recognition up to 60,000-fold. With the exception of Tyr and Phe, all residues are associated with comparable or greatly reduced catalytic activity relative to Pro. The crystal structures of three mutants that differ widely in catalytic activity (Y225F, Y225P, and Y225I) show that although residue 225 makes no contact with substrate, it drastically influences the shape of the water channel around the primary specificity site. The activity profiles obtained for thrombin also suggest that the conversion of Pro to Tyr or Phe documented in the vertebrates occurred through Ser and was driven by a significant gain (up to 50-fold) in catalytic activity. In fact, Ser and Phe are documented in 4% of serine proteases, which together with Pro and Tyr account for almost the entire distribution of residues at position 225. The unexpected crucial role of residue 225 in serine proteases explains the evolutionary selection of residues at this position and shows that the structural determinants of protease activity and specificity are more complex than currently believed. These findings have broad implications in the rational design of enzymes with enhanced catalytic properties.

A specific transition state for S-peptide combining with folded S-protein and then refolding

We measured the folding and unfolding kinetics of mutants for a simple protein folding reaction to characterize the structure of the transition state. Fluorescently labeled S-peptide analogues combine with S-protein to form ribonuclease S analogues: initially, S-peptide is disordered whereas S-protein is folded. The fluorescent probe provides a convenient spectroscopic probe for the reaction. The association rate constant, kon, and the dissociation rate constant, koff, were both determined for two sets of mutants. The dissociation rate constant is measured by adding an excess of unlabeled S-peptide analogue to a labeled complex (RNaseS*). This strategy allows kon and koff to be measured under identical conditions so that microscopic reversibility applies and the transition state is the same for unfolding and refolding. The first set of mutants tests the role of the α-helix in the transition state. Solvent-exposed residues Ala-6 and Gln-11 in the α-helix of native RNaseS were replaced by the helix destabilizing residues glycine or proline. A plot of log kon vs. log Kd for this series of mutants is linear over a very wide range, with a slope of −0.3, indicating that almost all of the molecules fold via a transition state involving the helix. A second set of mutants tests the role of side chains in the transition state. Three side chains were investigated: Phe-8, His-12, and Met-13, which are known to be important for binding S-peptide to S-protein and which also contribute strongly to the stability of RNaseS*. Only the side chain of Phe-8 contributes significantly, however, to the stability of the transition state. The results provide a remarkably clear description of a folding transition state.

Gonadotropin-releasing hormone analogue conjugates with strong selective antitumor activity

Conjugation of gonadotropin-releasing hormone (GnRH) analogues GnRH-III, MI-1544, and MI-1892 through lysyl side chains and a tetrapeptide spacer, Gly-Phe-Leu-Gly (X) to a copolymer, poly(N-vinylpyrrolidone-co-maleic acid) (P) caused increased antiproliferative activity toward MCF-7 and MDA-MB-231 breast, PC3 and LNCaP prostate, and Ishikawa endometrial cancer cell lines in culture and against tumor development by xenografts of the breast cancer cells in immunodeficient mice. MCF-7 cells treated with P-X-1544 and P-X-1892 displayed characteristic signs of apoptosis, including vacuoles in the cytoplasm, rounding up, apoptotic bodies, bleb formation, and DNA fragmentation. Conjugates, but not free peptides, inhibited cdc25 phosphatase and caused accumulation of Ishikawa and PC3 cells in the G2/M phase of the cell cycle after 24 h at lower doses and in the G1 and G2 phases after 48 h. Since P-X-peptides appear to be internalized, the increased cytotoxicity of the conjugates is attributed to protection of peptides from proteolysis, enhanced interaction of the peptides with the GnRH receptors, and/or internalization of P-X-peptide receptor complexes so that P can exert toxic effects inside, possibly by inhibiting enzymes involved in the cell cycle. The additional specificity of P-X-peptides compared with free peptides for direct antiproliferative effects on the cancer cells but not for interactions in the pituitary indicates the therapeutic potential of the conjugates.

Fidelity of G protein β-subunit association by the G protein γ-subunit-like domains of RGS6, RGS7, and RGS11

Several regulators of G protein signaling (RGS) proteins contain a G protein γ-subunit-like (GGL) domain, which, as we have shown, binds to Gβ5 subunits. Here, we extend our original findings by describing another GGL-domain-containing RGS, human RGS6. When RGS6 is coexpressed with different Gβ subunits, only RGS6 and Gβ5 interact. The expression of mRNA for RGS6 and Gβ5 in human tissues overlaps. Predictions of α-helical and coiled-coil character within GGL domains, coupled with measurements of Gβ binding by GGL domain mutants, support the contention that Gγ-like regions within RGS proteins interact with Gβ5 subunits in a fashion comparable to conventional Gβ/Gγ pairings. Mutation of the highly conserved Phe-61 residue of Gγ2 to tryptophan, the residue present in all GGL domains, increases the stability of the Gβ5/Gγ2 heterodimer, highlighting the importance of this residue to GGL/Gβ5 association.

Cerebral protein synthesis in a genetic mouse model of phenylketonuria

Local rates of cerebral protein synthesis (lCPSleu) were measured with the quantitative autoradiographic [1-14C]leucine method in a genetic mouse model (Pahenu2) of phenylketonuria. As in the human disease, Pahenu2 mice have a mutation in the gene for phenylalanine hydroxylase. We compared adult homozygous (HMZ) and heterozygous (HTZ) Pahenu2 mice with the background strain (BTBR). Arterial plasma concentrations of phenylalanine (Phe) were elevated in both HMZ and HTZ mutants by 21 times and 38%, respectively. In the total acid-soluble pool in brain concentrations of Phe were higher and other neutral amino acids lower in HMZ mice compared with either HTZ or BTBR mice indicating a partial saturation of the l-amino acid carrier at the blood brain barrier by the elevated plasma Phe concentrations. In a series of steady-state experiments, the contribution of leucine from the arterial plasma to the tRNA-bound pool in brain was found to be statistically significantly reduced in HMZ mice compared with the other groups, indicating that a greater fraction of leucine in the precursor pool for protein synthesis is derived from protein degradation. We found reductions in lCPSleu of about 20% throughout the brain in the HMZ mice compared with the other two groups, but no reductions in brain concentrations of tRNA-bound neutral amino acids. Our results in the mouse model suggest that in untreated phenylketonuria in adults, the partial saturation of the l-amino acid transporter at the blood–brain barrier may not underlie a reduction in cerebral protein synthesis.

Enhancing leptin response by preventing SH2-containing phosphatase 2 interaction with Ob receptor

Leptin is an adipocyte-derived cytokine that regulates food intake and body weight via interaction with its Ob receptor (ObR). Serum leptin levels are chronically elevated in obese humans, suggesting that obesity may be associated with leptin resistance and the inability to generate an adequate ObR response. Evidence suggests that transcriptional activation of target genes by STAT3 (signal transducer and activator of transcription) in the hypothalamus is a critical pathway that mediates leptin’s action. Herein we report that activation of ObR induces the tyrosine phosphorylation of the tyrosine phosphatase SH2-containing phosphatase 2 (SHP-2) and demonstrate that Tyr986 within the ObR cytoplasmic domain is essential to mediate phosphorylation of SHP-2 and binding of SHP-2 to ObR. Surprisingly, mutation of Tyr986 to Phe, which abrogates SHP-2 phosphorylation and binding to the receptor, dramatically increases gene induction mediated by STAT3. Our findings indicate that SHP-2 is a negative regulator of STAT3-mediated gene induction after activation of ObR and raise the possibility that blocking the interaction of SHP-2 with ObR could overcome leptin resistance by boosting leptin’s weight-reducing effects in obese individuals.

Visualization of elongation factor G on the Escherichia coli 70S ribosome: The mechanism of translocation

During protein synthesis, elongation factor G (EF-G) binds to the ribosome and promotes the step of translocation, a process in which tRNA moves from the A to the P site of the ribosome and the mRNA is advanced by one codon. By using three-dimensional cryo-electron microscopy, we have visualized EF-G in a ribosome–EF-G–GDP–fusidic acid complex. Fitting the crystal structure of EF-G–GDP into the cryo density map reveals a large conformational change mainly associated with domain IV, the domain that mimics the shape of the anticodon arm of the tRNA in the structurally homologous ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog. The tip portion of this domain is found in a position that overlaps the anticodon arm of the A-site tRNA, whose position in the ribosome is known from a study of the pretranslocational complex, implying that EF-G displaces the A-site tRNA to the P site by physical interaction with the anticodon arm.

Photochemical protease: Site-specific photocleavage of hen egg lysozyme and bovine serum albumin

Site-specific photocleavage of hen egg lysozyme and bovine serum albumin (BSA) by N-(l-phenylalanine)-4-(1-pyrene)butyramide (Py-Phe) is reported. Py-Phe binds to lysozyme and BSA with binding constants 2.2 ± 0.3 × 105 M−1 and 6.5 ± 0.4 × 107 M−1, respectively. Photocleavage of lysozyme and BSA was achieved with high specificity when a mixture of protein, Py-Phe, and an electron acceptor, cobalt(III) hexammine (CoHA), was irradiated at 344 nm. Quantum yields of photocleavage of lysozyme and BSA were 0.26 and 0.0021, respectively. No protein cleavage was observed in the absence of Py-Phe, CoHA, or light. N-terminal sequencing of the protein fragments indicated a single cleavage site of lysozyme between Trp-108 and Val-109, whereas the cleavage of BSA was found to be between Leu-346 and Arg-347. Laser flash photolysis studies of a mixture of protein, Py-Phe, and CoHA showed a strong transient with absorption centered at ≈460 nm, corresponding to pyrene cation radical. Quenching of the singlet excited state of Py-Phe by CoHA followed by the reaction of the resulting pyrenyl cation radical with the protein backbone may be responsible for the protein cleavage. The high specificity of photocleavage may be valuable in targeting specific sites of proteins with small molecules.

Quantitative assessment of enzyme specificity in vivo: P2 recognition by Kex2 protease defined in a genetic system

The specificity of the yeast proprotein-processing Kex2 protease was examined in vivo by using a sensitive, quantitative assay. A truncated prepro-α-factor gene encoding an α-factor precursor with a single α-factor repeat was constructed with restriction sites for cassette mutagenesis flanking the single Kex2 cleavage site (-SLDKR↓EAEA-). All of the 19 substitutions for the Lys (P2) residue in the cleavage site were made. The wild-type and mutant precursors were expressed in a yeast strain lacking the chromosomal genes encoding Kex2 and prepro-α-factor. Cleavage of the 20 sites by Kex2, expressed at the wild-type level, was assessed by using a quantitative-mating assay with an effective range greater than six orders of magnitude. All substitutions for Lys at P2 decreased mating, from 2-fold for Arg to >106-fold for Trp. Eviction of the Kex2-encoding plasmid indicated that cleavage of mutant sites by other cellular proteases was not a complicating factor. Mating efficiencies of strains expressing the mutant precursors correlated well with the specificity (kcat/KM) of purified Kex2 for comparable model peptide substrates, validating the in vivo approach as a quantitative method. The results support the conclusion that KM, which is heavily influenced by the nature of the P2 residue, is a major determinant of cleavage efficiency in vivo. P2 preference followed the rank order: Lys > Arg > Thr > Pro > Glu > Ile > Ser > Ala > Asn > Val > Cys > AsP > Gln > Gly > His > Met > Leu > Tyr > Phe > Trp.