Amino terminus of the interleukin-8 receptor is a major determinant of receptor subtype specificity.

Interleukin-8 (IL-8) is a key mediator in the migration of neutrophils from the circulation to the site of inflammation in the tissue. IL-8 is secreted by many cell types in response to proinflammatory stimuli such as interleukin 1, tumor necrosis factor, and lipopolysaccharide and is a potent chemoattractant and activator of neutrophils. Neutrophil activating peptide-2 (NAP-2) and melanoma growth-stimulatory activity (MGSA/GRO) are structurally and functionally related to IL-8 and, like IL-8, bind to specific G protein-coupled receptors on neutrophils. In the present study two closely related cloned IL-8 receptor subtypes are characterized by expression of the cDNA clones in monkey kidney cells (COS-7) or chinese hamster ovary cells and analysis of their ligand binding profiles. Both receptor subtypes bind 125I-labeled IL-8 with similar high affinity, however, the F3R receptor binds IL-8 exclusively, while the 4Ab receptor binds both IL-8 and MGSA/GRO with high affinity and NAP-2 with lesser affinity. Furthermore, we demonstrate with the use of intersubtype chimeric receptors that the specificity of ligand binding to both IL-8 receptor subtypes is dictated by the heterogeneous NH2-terminal domain. The F3R receptor is representative of a restricted IL-8 receptor subtype, and 4Ab represents a nonrestricted receptor subtype. It is proposed that these subtypes be named IL-8 receptors alpha and beta, respectively.     

A single amino acid can determine the DNA binding specificity of homeodomain proteins

Many Drosophila developmental genes contain a DNA binding domain encoded by the homeobox. This homeodomain contains a region distantly homologous to the helix-turn-helix motif present in several prokaryotic DNA binding proteins. We investigated the nature of homeodomain-DNA interactions by making a series of mutations in the helix-turn-helix motif of the Drosophila homeodomain protein Paired (Prd). This protein does not recognize sequences bound by the homeodomain proteins Fushi tarazu (Ftz) or Bicoid (Bcd). We show that changing a single amino acid at the C-terminus of the recognition helix is both necessary and sufficient to confer the DNA binding specificity of either Ftz or Bcd on Prd. This simple rule indicates that the amino acids that determine the specificity of homeodomains are different from those mediating protein-DNA contacts in prokaryotic proteins. We further show that Prd contains two DNA binding activities. The Prd homeodomain is responsible for one of them while the other is not dependent on the recognition helix.     

Species selectivity of nonpeptide antagonists of the gonadotropin-releasing hormone receptor is determined by residues in extracellular loops II and III and the amino terminus

Efforts to develop orally available gonadotropin-releasing hormone (GnRH) receptor antagonists have led to the discovery of several classes of potent nonpeptide antagonists. Here we investigated molecular interactions of three classes of nonpeptide antagonists with human, rat, and macaque GnRH receptors. Although all are high affinity ligands of the human receptor (K(i) <5 nm), these compounds show reduced affinity for the macaque receptor and bind only weakly (K(i) >1 microm) to the rat receptor. To identify residues responsible for this selectivity, a series of chimeric receptors and mutant receptors was constructed and evaluated for nonpeptide binding. Surprisingly, 4 key residues located in the amino terminus (Met-24) and extracellular loops II (Ser-203, Gln-208) and III (Leu-300) of the GnRH receptor appear to be primarily responsible for species-selective binding. Comparisons of reciprocal mutations suggest that these may not be direct contacts but rather may be involved in organizing extracellular portions of the receptor. These data are novel because most previous reports of residues involved in binding of nonpeptide ligands to peptide-activated G protein-coupled receptors, including the GnRH receptor as well as mono-amine receptors, have identified binding sites in the transmembrane regions.     

Analysis of the glucagon receptor first extracellular loop by the substituted cysteine accessibility method

Glucagon is an important hormone for the prevention of hypoglycemia, and contributes to the hyperglycemia observed in diabetic patients, yet very little is known about its receptor structure and the receptor-glucagon interaction. In related receptors, the first extracellular loop, ECL1, is highly variable in length and sequence, suggesting that it might participate in ligand recognition. We applied a variant of the SCAM (Substituted Cysteine Accessibility Method) to the glucagon receptor ECL1 and sequentially mutated positions 197 to 223 to cysteine. Most of the mutations (15/27) affected the glucagon potency, due either to a modification of the glucagon binding site, or to the destabilization of the active receptor conformation. We reasoned that side chains accessible to glucagon must also be accessible to large, hydrophilic cysteine reagents. We therefore evaluated the accessibility of the introduced cysteines to maleimide-PEO₂-biotin ((+)-biotinyl-3-maleimido-propionamidyl-3,6-dioxa-octanediamine), and tested the effect of pretreatment of intact cells with a large cationic cysteine reagent, MTSET ([2-(trimethylammonium)ethyl]methanethiosulfonate bromide), on glucagon potency. Our results suggest that the second and third transmembrane helices (TM2 and TM3) are extended to position 202 and from position 215, respectively, and separated by a short β stretch (positions 203-209). Glucagon binding induced a conformational change close to TM2: L198C was accessible to the biotin reagent only in the presence of glucagon. Most other mutations affected the receptor activation rather than glucagon recognition, but S217 and D218 (at the top of TM3) were good candidates for glucagon recognition and V221 was very close to the binding site.     

Three amino acid residues determine selective binding of FK506-binding protein 12.6 to the cardiac ryanodine receptor.

FK506-binding protein (FKBP12) has been found to be associated with the skeletal muscle ryanodine receptor (RyR1) (calcium release channel), whereas FKBP12.6, a novel isoform of FKBP, is selectively associated with the cardiac ryanodine receptor (RyR2). For both RyRs, the stoichiometry is 4 FKBP/RyR. Although FKBP12.6 differs from FKBP12 by only 18 of 108 amino acids, FKBP12.6 selectively binds to RyR2 and exchanges with bound FKBP12.6 of RyR2, whereas both FKBP isoforms bind to RyR1 and exchange with bound FKBP12 of RyR1. To assess the amino acid residues of FKBP12.6 that are critical for selective binding to RyR2, the residues of FKBP12.6 that differ with FKBP12 were mutated to the respective residues of FKBP12. RyR2 of cardiac sarcoplasmic reticulum, prelabeled by exchange with [35S]FKBP12.6, was used as assay system for binding/exchange with the mutants. The triple mutant (Q31E/N32D/F59W) of FKBP12.6 was found to lack selective binding to the cardiac RyR2, comparable with that of FKBP12.0. In complementary studies, mutations of FKBP12 to the three critical amino acids of FKBP12.6, conferred selective binding to RyR2. Each of the FKBP12.6 and FKBP12 mutants retained binding to the skeletal muscle RyR1. We conclude that three amino acid residues (Gln31, Asn32, and Phe59) of human FKBP12.6 account for the selective binding to cardiac RyR2.     

Structures of the intradiskal loops and amino terminus of the G-protein receptor, rhodopsin.

The intradiskal surface of the transmembrane protein, rhodopsin, consists of the amino terminal domain and three loops connecting six of the seven transmembrane helices. This surface corresponds to the extracellular surface of other G-protein receptors. Peptides that represent each of the extramembraneous domains on this surface (three loops and the amino terminus) were synthesized. These peptides also included residues which, based on a hydrophobic plot, could be expected to be part of the transmembrane helix. The structure of each of these peptides in solution was then determined using two-dimensional 1H nuclear magnetic resonance. All peptide domains showed ordered structures in solution. The structures of each of the peptides from intradiskal loops of rhodopsin exhibited a turn in the central region of the peptide. The ends of the peptides show an unwinding of the transmembrane helices to form this turn. The amino terminal domain peptide exhibited alpha-helical regions with breaks and bends at proline residues. This region forms a compact domain. Together, the structures for the loop and amino terminus domains indicate that the intradiskal surface of rhodopsin is ordered. These data further suggest a structural motif for short loops in transmembrane proteins. The ordered structures of these loops, in the absence of the transmembrane helices, indicate that the primary sequences of these loops are sufficient to code for the turn.     

Roles of specific extracellular domains of the glucagon receptor in ligand binding and signaling

To identify structural determinants of ligand binding in the glucagon receptor, eight receptor chimeras and additional receptor point mutants were prepared and studied. Amino acid residues 103-117 and 126-137 in the extracellular N-terminal tail and residues 206-219 and 220-231 in the first extracellular loop of the glucagon receptor were replaced with the corresponding segments of the glucagon-like peptide-1 receptor or the secretin receptor. Specific segments of both the N-terminal tail and the first extracellular loop of the glucagon receptor are required for hormone binding. The 206-219 segment of the first loop appears to be important for both glucagon binding and receptor activation. Functional studies with a synthetic chimeric peptide consisting of the N-terminal 14 residues of glucagon and the C-terminal 17 residues of glucagon-like peptide 1 suggest that hormone binding specificity may involve this segment of the first loop. The binding selectivity may arise in part from aspartic acid residues in this segment. Mutation of R-202 located at the junction between the second transmembrane helix and the first loop resulted in a mutant receptor that failed to bind glucagon or signal. We conclude that high-affinity glucagon binding requires multiple contacts with residues in the N-terminal tail and first extracellular loop domain of the glucagon receptor, with hormone specificity arising primarily from the amino acid 206-219 segment. The data suggest a model whereby glucagon first interacts with the N-terminal domain of the receptor followed by more specific interactions between the N-terminal half of the peptide and the first extracellular loop of the receptor, leading to activation.     

Role of extracellular charged amino acids in the yeast alpha-factor receptor

The yeast pheromone receptor, Ste2p, is a G protein coupled receptor that initiates cellular responses to alpha-mating pheromone, a 13 residue peptide that carries a net positive charge at physiological pH. We have examined the role of extracellular charged groups on the receptor in response to the pheromone. Substitutions of Asn or Ala for one extracellular residue, Asp275, affected both pheromone binding and signaling, suggesting that this position interacts directly with ligand. The other seven extracellular acidic residues could be individually replaced by polar residues with no detectable effects on receptor function. However, substitution of Ala for each of these seven residues resulted in impairment of signaling without affecting pheromone binding, implying that the polar nature of these residues promotes receptor activation. In contrast, substitution of Ala for each of the six positively charged residues at the extracellular surface of Ste2p did not affect signaling.     

The transmembrane topology of the amino terminus of the alpha subunit of the nicotinic acetylcholine receptor

We have investigated the transmembrane topology of the amino-terminal domain of the alpha subunit of the mouse muscle nicotinic acetylcholine receptor synthesized in vitro and in vivo. Using oligonucleotide-directed mutagenesis we introduced new glycosylation consensus sequences at alpha 154 and at alpha 200. For each novel site, additional constructs were made in which the original site at alpha N141 was eliminated. Glycosylation at the new sites, as exhibited in a rabbit reticulocyte cell-free translation system supplemented with canine pancreatic microsomes and in a transient transfection system with COS cells, was taken as evidence of the transmembrane translocation of the new site. Each of the new sites was glycosylated in both systems. In separate experiments we found that an alpha subunit fragment terminating at alpha M207 could be extracted from microsomal membranes with sodium carbonate after in vitro translation, indicating that this fragment is not an integral membrane protein. Our results, taken together with previous experiments, indicate that the amino terminus of the alpha subunit up to at least residue alpha 207 is translocated across the membrane of the endoplasmic reticulum. This topology probably represents the orientation of the amino terminus of the alpha subunit in the assembled receptor.     

Surface loops of extracellular phospholipase A(1) determine both substrate specificity and preference for lysophospholipids

Members of the pancreatic lipase family exhibit both lipase activity toward triacylglycerol and/or phospholipase A(1) (PLA(1)) activity toward certain phospholipids. Some members of the pancreatic lipase family exhibit lysophospholipase activity in addition to their lipase and PLA(1) activities. Two such enzymes, phosphatidylserine (PS)-specific PLA(1) (PS-PLA(1)) and phosphatidic acid (PA)-selective PLA(1)α (PA-PLA(1)α, also known as LIPH) specifically hydrolyze PS and PA, respectively. However, little is known about the mechanisms that determine their substrate specificities. Crystal structures of lipases and mutagenesis studies have suggested that three surface loops, namely, β5, β9, and lid, have roles in determining substrate specificity. To determine roles of these loop structures in the substrate recognition of these PLA(1) enzymes, we constructed a number of PS-PLA(1) mutants in which the three surface loops are replaced with those of PA-PLA(1)α. The results indicate that the surface loops, especially the β5 loop, of PA-PLA(1)α play important roles in the recognition of PA, whereas other structure(s) in PS-PLA(1) is responsible for PS preference. In addition, β5 loop of PS-PLA(1) has a crucial role in lysophospholipase activity toward lysophosphatidylserine. The present study revealed the critical role of lipase surface loops, especially the β5 loop, in determining substrate specificities of PLA(1) enzymes.     

Juxtamembrane region of the amino terminus of the corticotropin releasing factor receptor type 1 is important for ligand interaction

The functional properties of the amino terminus (NT) of the corticotropin releasing factor (CRF) receptor type 1 (R1) were studied by use of murine (m) CRFR1 and rat (r) parathyroid hormone (PTH)/parathyroid hormone-related peptide receptor (PTH1R) chimeras. The chimeric receptor CXP, in which the NT of mCRFR1 was annealed to the TMs of PTH1R, and the reciprocal hybrid, PXC, bound radiolabeled analogues of sauvagine and PTH(3--34), respectively. Neither hybrid bound radiolabeled CRF or PTH(1--34). CRF and PTH(1--34) weakly stimulated intracellular cAMP accumulation in COS-7 cells transfected with PXC and CXP, respectively. Thus the NT is required for ligand binding and the TMs are required for agonist-stimulated cAMP accumulation. Replacing individual intercysteine segments of PXC with their mCRFR1 counterparts did not rescue CRF or sauvagine radioligand binding or stimulation of cAMP accumulation. Replacement of residues 1--31 of mCRFR1 with their PTH1R counterparts resulted in a chimeric receptor, PEC, which had normal CRFR1 functional properties. In addition, a series of chimeras (F1PEC--F6PEC) were generated by replacement of the NT intercysteine residues of PEC with their PTH1R counterparts. Only F1PEC, F2PEC, and F3PEC showed detectable CRF and sauvagine radioligand binding. All of the PEC chimeras except F5PEC increased cAMP accumulation. These data indicate that the Cys(68)(-)Glu(109) domain is important for binding and that the Cys(87)(-)Cys(102) region plays an important role in CRFR1 activation.     

Prostaglandin moieties that determine receptor binding specificity in the bovine corpus luteum.

This study provided a pharmacological evaluation of prostaglandin binding to bovine luteal plasma membrane. It was found that [3H]PGF2 alpha' [3H]PGE2' [3H]PGE1 and [3H]PGD2 all bound with high affinity to luteal plasma membrane but had different specificities. Binding of [3H]PGF2 alpha and [3H]PGD2 was inhibited by non-radioactive PGF2 alpha (IC50 values of 21 and 9 nmol l-1, respectively), PGD2 (35 and 21 nmol l-1), and PGE2 (223 and 81 nmol l-1), but not by PGE1 (> 10,000 and 5616 nmol l-1). In contrast, [3H]PGE1 was inhibited by non-radioactive PGE1 (14 nmol l-1) and PGE2 (7 nmol l-1), but minimally by PGD2 (2316 nmol l-1) and PGF2 alpha (595 nmol l-1). Binding of [3H]PGE2 was inhibited by all four prostaglandins, but slopes of the dissociation curves indicated two binding sites. Binding of [3H]PGE1 was inhibited, resulting in low IC50 values, by pharmacological agonists that are specific for EP3 receptor and possibly EP2 receptor. High affinity binding of [3H]PGF2 alpha required a C15 hydroxyl group and a C1 carboxylic acid that are present on all physiological prostaglandins. Specificity of binding for the FP receptor depended on the C9 hydroxyl group and the C5/C6 double bond. Alteration of the C11 position had little effect on affinity for the FP receptor. In conclusion, there is a luteal EP receptor with high affinity for PGE1' PGE2' agonists of EP3 receptors, and some agonists of EP2 receptors. The luteal FP receptor binds PGF2 alpha' PGD2 (high affinity), and PGE2 (moderate affinity) but not PGE1 due to affinity determination by the C9 and C5/C6 moieties, but not the C11 moiety.     

Three distinct epitopes within the loop region of hen egg lysozyme defined with monoclonal antibodies.

Five monoclonal antibodies specific for the loop region of hen egg lysozyme were prepared by immunisation with a synthetic conjugate of a proteolytic fragment of lysozyme coupled to bovine serum albumin. Their fine specificities were investigated using a panel of variant lysozymes and peptide fragments of lysozyme in a quantitative radio-immunoassay procedure. Knowledge of the structure of hen lysozyme to high resolution and the use of computer graphics enables the localisation of the epitopes recognised by the antibodies with some precision. The antibodies were shown to define three distinct, overlapping epitopes within what was previously considered to be a single antigenic site. These results are discussed in relation to current ideas of the antigenic nature of proteins and other recent studies in which anti-protein antibodies have been elicited by immunisation with small peptides.     

Predicted complementarity determining regions of the T cell antigen receptor determine antigen specificity.

The antigen receptor on T cells (TCR) has been predicted to have a structure similar to a membrane-anchored form of an immunoglobulin F(ab) fragment. Virtually all of the conserved amino acids that are important for inter- and intramolecular interactions in the VH-VL pair are also conserved in the TCR V alpha and V beta chains. A molecular model of the TCR has been constructed by homology and we have used the information from this, as well as the earlier structural predictions of others, to study the basis for specificity. Specifically, regions of a TCR cloned from an antigen-specific T cell were stitched into the corresponding framework of a second TCR. Results indicate that the substitution of amino acid sequences corresponding to the complementarity determining regions (CDRs) of immunoglobulin can convey the specificity for antigen and major histocompatibility complex molecules. These data are consistent with a role, but not an exclusive role, for CDR3 in antigen peptide recognition.     

Noncontiguous regions in the extracellular domain of EGF receptor define ligand-binding specificity.

Murine epidermal growth factor (EGF) binds with approximately 250-fold higher binding affinity to the human EGF receptor (EGFR) than to the chicken EGFR. This difference in binding affinity enabled the identification of a major ligand-binding domain for EGF by studying the binding properties of various chicken/human EGFR chimera expressed in transfected cells lacking endogenous EGFR. It was shown that domain III of EGFR is a major ligand-binding region. Here, we analyze the binding properties of novel chicken/human chimera to further delineate the contact sequences in domain III and to assess the role of other regions of EGFR for their contribution to the display of high-affinity EGF binding. The chimeric receptors include chicken EGFR containing domain I of the human EGFR, chicken receptor containing domain I and III of the human EGFR, and two chimeric chicken EGFR containing either the amino terminal or the carboxy terminal halves of domain III of human EGFR, respectively. In addition, the binding of various human-specific anti-EGFR monoclonal antibodies that interfere with EGF binding is also compared. It is concluded that noncontiguous regions of the EGFR contribute additively to the binding of EGF. Each of the two halves of domain III has a similar contribution to the binding energy, and the sum of both is close to that of the entire domain III. This suggests that the folding of domain III juxtaposes sequences that together constitute the ligand-binding site. Domain I also provides a contribution to the binding energy, and the added contributions of both domain I and III to the binding energy generate the high-affinity binding site typical of human EGFR.     

Tau polymerization: role of the amino terminus

The abnormal polymerization of the tau molecule into insoluble filaments is a seminal event in the neurodegenerative process underlying Alzheimer's disease. Previous experimentation has shown that the microtubule-binding repeat region of the molecule is vital for its ability to polymerize in vitro into filaments similar to those found in Alzheimer's disease. However, it is becoming clear that regions outside the microtubule-binding repeat, such as exons 2 and 3 and the carboxy-terminal tail, can greatly influence its polymerization. Since it has been previously postulated that the amino terminus of tau could be involved in generating pathological conformations in the disease state, its role in the polymerization process was investigated. This report demonstrates that the removal of the amino terminus greatly inhibits the polymerization of the tau molecule, reducing both the rate and extent of polymerization. These results support the hypothesis that the ability of tau to form specific conformations involving the amino terminus is an early event in the formation of tau polymers in the disease state. Furthermore, the mutation of arginine 5 to leucine ((R)5(L)), mimicking an amino-terminal tau mutation found in a single case of FTDP-17, enhances the polymerization of the tau molecule. Therefore, the amino terminus of the tau molecule, while largely overlooked in studies of its polymerization, is a significant contributor to the polymerization process.