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1.
J Biol Chem ; 276(5): 3394-400, 2001 Feb 02.
Article in English | MEDLINE | ID: mdl-11062244

ABSTRACT

Although agonists are thought to occupy binding pockets within the seven-helix core of serpentine receptors, the topography of these binding pockets and the conformational changes responsible for receptor activation are poorly understood. To identify the ligand binding pocket in the receptor for complement factor 5a (C5aR), we assessed binding affinities of hexapeptide ligands, each mutated at a single position, for seven mutant C5aRs, each mutated at a single position in the putative ligand binding site. In ChaW (an antagonist) and W5Cha (an agonist), the side chains at position 5 are tryptophan and cyclohexylalanine, respectively. Comparisons of binding affinities indicated that the hexapeptide residue at this position interacts with two C5aR residues, Ile-116 (helix III) and Val-286 (helix VII); in a C5aR model these two side chains point toward one another. Both the I116A and the V286A mutations markedly increased binding affinity of W5Cha but not that of ChaW. Moreover, ChaW, the antagonist hexapeptide, acted as a full agonist on the I116A mutant. These results argue that C5aR residues Ile-116 and Val-286 interact with the side chain at position 5 of the hexapeptide ligand to form an activation switch. Based on this and previous work, we present a docking model for the hexapeptide within the C5aR binding pocket. We propose that agonists induce a small change in the relative orientations of helices III and VII and that these helices work together to allow movement of helix VI away from the receptor core, thereby triggering G protein activation.


Subject(s)
Antigens, CD/chemistry , Receptors, Complement/chemistry , Signal Transduction/physiology , Alanine/genetics , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Binding Sites , COS Cells , Isoleucine/genetics , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Mutation , Peptides/chemistry , Protein Conformation , Receptor, Anaphylatoxin C5a , Receptors, Complement/genetics , Receptors, Complement/metabolism
2.
Circ Res ; 87(9): 753-9, 2000 Oct 27.
Article in English | MEDLINE | ID: mdl-11055978

ABSTRACT

On the basis of the patterns of conserved amino acid sequence, the angiotensin II type 2 (AT(2)) receptor belongs to the family of serpentine receptors, which relay signals from extracellular stimuli to heterotrimeric G proteins. However, the AT(2) receptor signal transduction mechanisms are poorly understood. We have measured AT(2)-triggered activation of purified heterotrimeric proteins in urea-extracted membranes from cultured COS-7 cells expressing the recombinant receptor. This procedure removes contaminating GTP-binding proteins without inactivating the serpentine receptor. Binding studies using [(125)I] angiotensin (Ang) II revealed a single binding site with a K(d)=0.45 and a capacity of 627 fmol/mg protein in the extracted membranes. The AT(2) receptor caused a rapid activation of alpha(i) and alpha(o) but not of alpha(q) and alpha(s), as measured by radioactive guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding. Activation required the presence of activated receptors, betagamma, and alpha subunits. As a first step aimed at developing an in vitro assay to examine AT(2) receptor pharmacology, we tested a battery of Ang II-related ligands for their ability to promote AT(1) or AT(2) receptor-catalyzed G(i) activation. Two proteolytic fragments of Ang II, Ang III and Ang1-7, also promoted activation of alpha(i) through the AT(2) receptor. Furthermore, we found that [Sar(1),Ala(8)]Ang II is an antagonist for both AT(1) and AT(2) receptors and that CPG42112 behaves as a partial agonist for the AT(2) receptor. In combination with previous observations, these results show that the AT(2) receptor is fully capable of activating G(i) and provides a new tool for exploring AT(2) receptor pharmacology and interactions with G-protein trimers.


Subject(s)
GTP-Binding Proteins/metabolism , Receptors, Angiotensin/metabolism , 3T3 Cells , Angiotensin II/chemistry , Angiotensin II/metabolism , Animals , Binding Sites , COS Cells , Cell Membrane/metabolism , GTP-Binding Proteins/analysis , Gene Expression , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Kinetics , Ligands , Mice , Peptide Fragments/metabolism , Point Mutation , Receptor, Angiotensin, Type 1 , Receptor, Angiotensin, Type 2 , Receptors, Angiotensin/drug effects , Receptors, Angiotensin/genetics , Signal Transduction , Structure-Activity Relationship , Transfection , Urea
3.
J Biol Chem ; 275(45): 35393-401, 2000 Nov 10.
Article in English | MEDLINE | ID: mdl-10952985

ABSTRACT

Many hormones and sensory stimuli signal through a superfamily of seven transmembrane-spanning receptors to activate heterotrimeric G proteins. How the seven transmembrane segments of the receptors (a molecular architecture of bundled alpha-helices conserved from yeast to man) work as "on/off" switches remains unknown. Previously, we used random saturation mutagenesis coupled with a genetic selection in yeast to determine the relative importance of amino acids in four of the seven transmembrane segments of the human C5a receptor (Baranski, T. J., Herzmark, P., Lichtarge, O., Gerber, B. O., Trueheart, J., Meng, E. C., Iiri, T., Sheikh, S. P., and Bourne, H. R. (1999) J. Biol. Chem. 274, 15757-15765). In this study, we evaluate helices I, II, and IV, thereby furnishing a complete mutational map of the seven transmembrane helices of the human C5a receptor. Our analysis identified 19 amino acid positions resistant to non-conservative substitutions. When combined with the 25 essential residues previously identified in helices III and V-VII, they delineate two distinct components of the receptor switch: a ligand-binding surface at or near the extracellular surface of the helix bundle and a core cluster in the cytoplasmic half of the bundle. In addition, we found critical amino acids in the first and second helices that are predicted to face the lipid membrane. These residues form an extended surface that might mediate interactions with lipids and other membrane proteins or function as an oligomerization domain with other receptors.


Subject(s)
Antigens, CD/chemistry , Antigens, CD/genetics , Receptors, Complement/chemistry , Receptors, Complement/genetics , Amino Acid Sequence , Amino Acids/chemistry , Antigens, CD/physiology , Cell Membrane/chemistry , Chromosome Mapping , Fungal Proteins , Gene Library , Genotype , Humans , Ligands , Models, Biological , Models, Molecular , Molecular Sequence Data , Mutagenesis , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Receptor, Anaphylatoxin C5a , Receptors, Complement/physiology , Recombination, Genetic , Sequence Homology, Amino Acid
4.
J Biol Chem ; 274(24): 17033-41, 1999 Jun 11.
Article in English | MEDLINE | ID: mdl-10358054

ABSTRACT

The seven transmembrane helices of serpentine receptors comprise a conserved switch that relays signals from extracellular stimuli to heterotrimeric G proteins on the cytoplasmic face of the membrane. By substituting histidines for residues at the cytoplasmic ends of helices III and VI in retinal rhodopsin, we engineered a metal-binding site whose occupancy by Zn(II) prevented the receptor from activating a retinal G protein, Gt (Sheikh, S. P., Zvyaga, T. A. , Lichtarge, O., Sakmar, T. P., and Bourne, H. R. (1996) Nature 383, 347-350). Now we report engineering of metal-binding sites bridging the cytoplasmic ends of these two helices in two other serpentine receptors, the beta2-adrenoreceptor and the parathyroid hormone receptor; occupancy of the metal-binding site by Zn(II) markedly impairs the ability of each receptor to mediate ligand-dependent activation of Gs, the stimulatory regulator of adenylyl cyclase. We infer that these two receptors share with rhodopsin a common three-dimensional architecture and an activation switch that requires movement, relative to one another, of helices III and VI; these inferences are surprising in the case of the parathyroid hormone receptor, a receptor that contains seven stretches of hydrophobic sequence but whose amino acid sequence otherwise shows no apparent similarity to those of receptors in the rhodopsin family. These findings highlight the evolutionary conservation of the switch mechanism of serpentine receptors and help to constrain models of how the switch works.


Subject(s)
Evolution, Molecular , Receptors, Adrenergic, beta-2/metabolism , Receptors, Parathyroid Hormone/metabolism , Zinc/pharmacology , Animals , Binding, Competitive , GTP-Binding Protein alpha Subunits, Gs/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Histidine/genetics , Humans , Isoproterenol/metabolism , Models, Molecular , Mutation , Pindolol/analogs & derivatives , Pindolol/metabolism , Protein Engineering , Protein Structure, Secondary , Receptors, Adrenergic, beta-2/chemistry , Receptors, Adrenergic, beta-2/drug effects , Receptors, Adrenergic, beta-2/genetics , Receptors, Parathyroid Hormone/chemistry , Receptors, Parathyroid Hormone/drug effects , Receptors, Parathyroid Hormone/genetics , Rod Opsins , Secretin , Sequence Alignment , Signal Transduction
5.
J Biol Chem ; 274(22): 15757-65, 1999 May 28.
Article in English | MEDLINE | ID: mdl-10336477

ABSTRACT

Hormones and sensory stimuli activate serpentine receptors, transmembrane switches that relay signals to heterotrimeric guanine nucleotide-binding proteins (G proteins). To understand the switch mechanism, we subjected 93 amino acids in transmembrane helices III, V, VI, and VII of the human chemoattractant C5a receptor to random saturation mutagenesis. A yeast selection identified 121 functioning mutant receptors, containing a total of 523 amino acid substitutions. Conserved hydrophobic residues are located on helix surfaces that face other helices in a modeled seven-helix bundle (Baldwin, J. M., Schertler, G. F., and Unger, V. M. (1997) J. Mol. Biol. 272, 144-164), whereas surfaces predicted to contact the surrounding lipid tolerate many substitutions. Our analysis identified 25 amino acid positions resistant to nonconservative substitutions. These appear to comprise two distinct components of the receptor switch, a surface at or near the extracellular membrane interface and a core cluster in the cytoplasmic half of the bundle. Twenty-one of the 121 mutant receptors exhibit constitutive activity. Amino acids substitutions in these activated receptors predominate in helices III and VI; other activating mutations truncate the receptor near the extracellular end of helix VI. These results identify key elements of a general mechanism for the serpentine receptor switch.


Subject(s)
Antigens, CD/chemistry , Membrane Proteins/genetics , Protein Structure, Secondary , Receptors, Complement/chemistry , Amino Acids/genetics , Antigens, CD/genetics , Complement C5a/metabolism , Evolution, Molecular , Gene Library , Humans , Membrane Proteins/chemistry , Models, Molecular , Mutation , Receptor, Anaphylatoxin C5a , Receptors, Complement/genetics , Sequence Deletion , Signal Transduction , Yeasts/genetics
6.
Proc Natl Acad Sci U S A ; 96(2): 499-504, 1999 Jan 19.
Article in English | MEDLINE | ID: mdl-9892662

ABSTRACT

Hormonal signals activate trimeric G proteins by substituting GTP for GDP bound to the G protein alpha subunit (Galpha), thereby generating two potential signaling molecules, Galpha-GTP and free Gbetagamma. The usefulness of dominant negative mutations for investigating Ras and other monomeric G proteins inspired us to create a functionally analogous dominant negative Galpha mutation. Here we describe a mutant alpha subunit designed to inhibit receptor-mediated hormonal activation of Gs, the stimulatory regulator of adenylyl cyclase. To construct this mutant, we introduced into the alpha subunit (alphas) of Gs three separate mutations chosen because they impair alphas function in complementary ways: the A366S mutant reduces affinity of alphas for binding GDP, whereas the G226A and E268A mutations impair the protein's ability to bind GTP and to assume an active conformation. The triple mutant robustly inhibits (by up to 80%) Gs-dependent hormonal stimulation of adenylyl cyclase in cultured cells. Inhibition is selective in that it does not affect cellular responses to expression of a constitutively active alphas mutant (alphas-R201C) or to agonists for receptors that activate Gq or Gi. This alphas triple mutant and cognate Galpha mutants should provide specific tools for dissection of G protein-mediated signals in cultured cells and transgenic animals.


Subject(s)
GTP-Binding Proteins/genetics , Mitogen-Activated Protein Kinases , Mutation/genetics , Signal Transduction/genetics , Adenylyl Cyclase Inhibitors , Animals , COS Cells , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Chorionic Gonadotropin/pharmacology , Cyclic AMP/metabolism , Mitogen-Activated Protein Kinase 3 , Protein Binding/genetics , Receptors, Cell Surface/antagonists & inhibitors , Receptors, LH/genetics , Transfection/genetics
7.
J Biol Chem ; 270(1): 170-9, 1995 Jan 06.
Article in English | MEDLINE | ID: mdl-7814370

ABSTRACT

A novel combinatorial mutagenesis strategy (shuffle mutagenesis) was developed to identify sequences in the propiece and amino lobe of cathepsin D which direct oligosaccharide phosphorylation by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine 1-phosphotransferase. Propiece restriction fragments and oligonucleotide cassettes corresponding to 13 regions of the cathepsin D and glycopepsinogen amino lobes were randomly shuffled together to generate a large library of chimeric molecules. The library was inserted into an expression vector encoding the carboxyl lobe of cathepsin D with a carboxyl-terminal myc epitope and a CD8 transmembrane extension. Transfected COS1 cells expressing the membrane-anchored forms of the cathepsin D/glycopepsinogen chimeras at the cell surface were selected with solid phase mannose 6-phosphate receptor or an antibody to the myc epitope. Plasmids were rescued in Escherichia coli and sequenced by hybridization to the original oligonucleotide cassettes. Two regions of the cathepsin D amino lobe (segments 7 and 12) were found to contribute to proper folding, surface expression, and selective phosphorylation of the carboxyl lobe oligosaccharide. Two different cathepsin D regions (the propiece and segment 5) cooperated with a previously identified recognition element in the carboxyl lobe to allow efficient phosphorylation of both the amino and carboxyl lobe oligosaccharides. Three general models for extending the catalytic reach of N-acetylglucosamine 1-phosphotransferase to widely spaced oligosaccharides are presented.


Subject(s)
Cathepsin D/metabolism , Enzyme Precursors/metabolism , Mutagenesis , Oligosaccharides/metabolism , Transferases (Other Substituted Phosphate Groups)/metabolism , Amino Acid Sequence , Animals , Aspartic Acid Endopeptidases/genetics , Cathepsin D/genetics , Cell Line , Cell Membrane/enzymology , Enzyme Precursors/genetics , Genetic Vectors , Molecular Sequence Data , Phosphorylation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transferases (Other Substituted Phosphate Groups)/genetics
8.
J Biol Chem ; 267(32): 23342-8, 1992 Nov 15.
Article in English | MEDLINE | ID: mdl-1331081

ABSTRACT

We have investigated the nature of a protein domain that is shared among lysosomal hydrolases and is recognized by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the biosynthesis of mannose 6-phosphate residues. Previously, elements of this recognition domain were identified using a chimeric protein approach. The combined substitution of two regions (amino acids 188-230, particularly lysine 203, and 265-292) from the carboxyl lobe of the lysosomal hydrolase cathepsin D into the homologous positions of the related secretory protein glycopepsinogen was sufficient to confer recognition by phosphotransferase and subsequent phosphorylation of the oligosaccharides when this chimeric protein was expressed in Xenopus oocytes. (Baranski, T. J., Faust, P. L., and Kornfeld, S. (1990) Cell 63, 281-291). The current study demonstrates that when these two regions are replaced in cathepsin D by the homologous glycopepsinogen amino acids, the resultant chimeric molecule is poorly phosphorylated. However, when either of these regions is substituted individually, the chimeric molecules are well phosphorylated. The phosphorylation of these latter chimeric proteins is dependent on the presence of procathepsin D amino lobe elements. By analyzing a series of chimeric proteins that contain all eight combinations of three consecutive segments of the entire amino lobe of procathepsin D, it was found that multiple regions of the amino lobe of cathepsin D enhance phosphorylation of the chimeric proteins. These elements may be part of an extended carboxyl lobe recognition domain or comprise a second independent recognition domain.


Subject(s)
Cathepsin D/metabolism , Enzyme Precursors/metabolism , Lysosomes/enzymology , Phosphotransferases/metabolism , Transferases (Other Substituted Phosphate Groups) , Amino Acid Sequence , Animals , Cathepsin D/genetics , Cathepsin D/isolation & purification , Chromatography, Affinity , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Cloning, Molecular , DNA/genetics , Enzyme Precursors/genetics , Enzyme Precursors/isolation & purification , Glycopeptides/isolation & purification , Humans , Lysine , Mutagenesis, Site-Directed , Oligosaccharides/isolation & purification , Oocytes/enzymology , Peptide Mapping , Phosphorylation , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Restriction Mapping , Transcription, Genetic , Xenopus
9.
J Biol Chem ; 267(32): 23349-56, 1992 Nov 15.
Article in English | MEDLINE | ID: mdl-1331082

ABSTRACT

Cathepsin D is a bilobed lysosomal aspartyl protease that contains one Asn-linked oligosaccharide/lobe. Each lobe also contains protein determinants that serve as recognition domains for binding of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the first enzyme in the biosynthesis of the mannose 6-phosphate residues on lysosomal enzymes. In this study we examined whether the location of the protein recognition domain influences the relative phosphorylation of the amino and carboxyl lobe oligosaccharides. To do this, chimeric proteins containing either amino or carboxyl lobe sequences of cathepsin D substituted into a glycosylated form of the homologous secretory protein pepsinogen were expressed in Xenopus oocytes. The amino and carboxyl lobe oligosaccharides were then isolated from the various chimeric proteins and independently analyzed for their mannose 6-phosphate content. This analysis has shown that a phosphotransferase recognition domain located on either lobe of a cathepsin D/glycopepsinogen chimeric molecule is sufficient to allow phosphorylation of oligosaccharides on both lobes. However, phosphorylation of the oligosaccharide on the lobe containing the recognition domain is favored. We also found that the majority of the carboxyl lobe oligosaccharides of cathepsin D acquire two phosphates, whereas the amino lobe oligosaccharides only acquire one phosphate.


Subject(s)
Cathepsin D/genetics , Cathepsin D/metabolism , Enzyme Precursors/genetics , Enzyme Precursors/metabolism , Lysosomes/enzymology , Oligosaccharides/metabolism , Phosphotransferases/metabolism , Transferases (Other Substituted Phosphate Groups) , Animals , Base Sequence , Binding Sites , Carbohydrate Conformation , Carbohydrate Sequence , Chromatography, Affinity , Chromatography, Ion Exchange , Cloning, Molecular , Female , Glycopeptides/biosynthesis , Glycopeptides/isolation & purification , Humans , Kidney/enzymology , Mannose/metabolism , Models, Molecular , Molecular Sequence Data , Oligodeoxyribonucleotides , Pepsinogens/genetics , Pepsinogens/metabolism , Phosphorylation , Plasmids , Polymerase Chain Reaction , Protein Conformation , Xenopus laevis
10.
J Biol Chem ; 266(34): 23365-72, 1991 Dec 05.
Article in English | MEDLINE | ID: mdl-1660471

ABSTRACT

Lysosomal enzymes contain a common protein determinant that is recognized by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the biosynthesis of mannose-6-P residues. Previously, we generated a lysosomal enzyme recognition domain by substituting two regions (lysine 203 and amino acids 265-292) of the lysosomal hydrolase cathepsin D into a related secretory protein glycopepsinogen. When expressed in Xenopus oocytes, the oligosaccharides of the chimeric protein were efficiently phosphorylated (Baranski, T. J., Faust, P. L., and Kornfeld, S. (1990) Cell 63, 281-291). In the current study, incremental substitutions of cathepsin D residues into glycopepsinogen and alanine-scanning mutagenesis were utilized to define the recognition domain more precisely. A computer-generated model of the cathepsin D/pepsinogen chimeric molecule served as a guide for mutagenesis and for the interpretation of results. These studies indicate that the recognition domain is a surface patch that contains multiple interacting sites. There is a strict positional requirement for the lysine residue at position 203.


Subject(s)
Cathepsin D/metabolism , Lysosomes/enzymology , Phosphotransferases/metabolism , Transferases (Other Substituted Phosphate Groups) , Amino Acid Sequence , Animals , Binding Sites , Cathepsin D/chemistry , Computer Simulation , Humans , Models, Molecular , Molecular Sequence Data , Mutagenesis , Sequence Alignment , Swine
11.
Plant Cell ; 2(11): 1091-1106, 1990 Nov.
Article in English | MEDLINE | ID: mdl-12354950

ABSTRACT

Aleurain, originally described from its cDNA as a thiol protease [Rogers, J.C., Dean, D., and Heck, G.R. (1985). Proc. Natl. Acad. Sci. USA 82, 6512-6516], is characterized here as a glycoprotein that is targeted to a distinct vacuolar compartment in aleurone cells. Monospecific antibodies to a bacterial trpE-aleurain fusion protein were used to show that aleurain is made as a 42-kilodalton (kD) proenzyme (proaleurain) that is proteolytically processed in a post-Golgi compartment in two steps to form a 32-kD protein. The first processing step is the discrete loss of 9 kD from proaleurain to yield a 33-kD intermediate that is further processed by the gradual loss of 1 kD resulting in mature 32-kD aleurain. Using proaleurain secreted from Xenopus oocytes as a substrate, we established an in vitro system using aleurone cell extracts that correctly processes proaleurain to a stable protein that is indistinguishable from native barley aleurain as judged by partial digestion with staphylococcal V8 protease. Proaleurain is not capable of self-cleavage in the absence of aleurone cell extracts and mature aleurain appears not to participate in processing in vitro.

12.
Cell ; 63(2): 281-91, 1990 Oct 19.
Article in English | MEDLINE | ID: mdl-2170024

ABSTRACT

Lysosomal enzymes contain a common protein determinant that is recognized by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the formation of mannose 6-phosphate residues. To identify this protein determinant, we constructed chimeric molecules between two aspartyl proteases: cathepsin D, a lysosomal enzyme, and pepsinogen, a secretory protein. When expressed in Xenopus oocytes, the oligosaccharides of cathepsin D were efficiently phosphorylated, whereas the oligosaccharides of a glycosylated form of pepsinogen were not phosphorylated. The combined substitution of two noncontinuous sequences of cathepsin D (lysine 203 and amino acids 265-292) into the analogous positions of glycopepsinogen resulted in phosphorylation of the oligosaccharides of the expressed chimeric molecule. These two sequences are in direct apposition on the surface of the molecule, indicating that amino acids from different regions come together in three-dimensional space to form this recognition domain. Other regions of cathepsin D were identified that may be components of a more extensive recognition marker.


Subject(s)
Lysosomes/enzymology , Pepsinogens/genetics , Pepsinogens/metabolism , Phosphotransferases/metabolism , Transferases (Other Substituted Phosphate Groups) , Amino Acid Sequence , Animals , Base Sequence , Cathepsin D/genetics , Chromatography, Affinity , Cloning, Molecular , Humans , Lysine , Mannosephosphates/metabolism , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligonucleotide Probes , Oligosaccharides/isolation & purification , Oocytes/metabolism , Phosphorylation , Protein Conformation , Receptor, IGF Type 2 , Receptors, Cell Surface/metabolism , Sequence Homology, Nucleic Acid , Transcription, Genetic , Xenopus laevis
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