Importance of extracellular domains for ligand binding in the thyrotropin-releasing hormone receptor

The role of putative extracellular sequences for ligand binding in the TRH receptor was examined using deletion or substitution mutations. Each mutant receptor was transiently expressed in TRH receptor-minus GH(1)2C(1)b rat pituitary cells, and binding of 4 Nu Mu [3H]pGlu-N(tau)-MeHis-Pro-NH2 ([3H] MeTRH) was measured. When binding was not detected, signal transduction at 10 microM MeTRH was measured to assess receptor expression. Deletion of most of the N-terminal sequences (Glu(2)-Leu(22)), including two potential glycosylation sites, had no effect on the affinity of the receptor for MeTRH. Segmental deletions or simultaneous substitution of multiple amino acid residues in the first, second, or third extracellular loop (EL1, EL2, or EL3) resulted, however, in total loss of [3H]MeTRH binding, suggesting important roles for the loop sequences in either receptor expression or ligand binding. Individual substitutions were made to test further the role of the specific extracellular loop sequences in TRH binding. In EL1, conversion of Tyr93 to Ala resulted in more than 20-fold decrease in affinity for MeTRH. In EL2 and the top portion of the fifth transmembrane helix, conversion of Tyr181 to Phe, Tyr188 to Ala, and Phe199 to Ala resulted in a large ( > 100-fold) decrease in affinity for MeTRH, and conversion of Tyr 188 to Phe and Phe196 to Ala caused an agonist-specific 4- to 5-fold decrease in affinity. In EL3, conversion of Asn289 to Ala and of Ser290 to Ala caused a large ( > 100-fold) decrease in affinity for MeTRH. These results suggest important roles for the extracellular loops in high affinity TRH binding and lead us to propose a model in which TRH binds to the extra-cellular domain of its receptor.     

Characterization of the extracellular domain in vascular endothelial growth factor receptor-1 (Flt-1 tyrosine kinase)

Flt-1 tyrosine kinase, vascular endothelial growth factor (VEGF) receptor-1, binds VEGF and a new VEGF-related ligand, placenta growth factor, but KDR/Flk-1 (VEGF receptor-2) binds only VEGF. To characterize the functional regions in the Flt-1 extracellular domain such as the ligand binding region and the dimer formation of the receptor, we constructed a series of mutants of the Flt-1 extracellular domain as soluble forms in a baculovirus system. We found that a region carrying the N-terminal 1st to 3rd immunoglobulin (Ig)-like domains of Flt-1 binds both ligands with high affinity. However, for dimer formation of soluble Flt-1, a region further downstream in the Flt-1 extracellular domain was required. Mutant Flt-1 receptors expressed in COS cells confirmed the requirement of the 4th to 7th Ig region for the activation of Flt-1 tyrosine kinase. Soluble Flt-1 carrying the N-terminal 1st to 3rd Ig region suppressed VEGF-dependent endothelial proliferation in vitro to the same level as the larger forms of soluble Flt-1, suggesting that the binding of one soluble Flt-1 molecule to one subunit of the VEGF homodimer may be sufficient to block the VEGF activity.     

Evidence indicating that the extracellular loops of the mouse MC5 receptor do not participate in ligand binding

Inulin ethers carrying primary amino groups have many potential applications. O-(Aminopropyl)inulin is obtained from O-(cyanoethyl)inulin by reduction of the nitrile groups. Heterogeneously catalyzed hydrogenation using Raney-cobalt as the catalyst resulted in only partial conversion of the O-cyanoethyl into O-aminopropyl groups. Complete conversion of the nitriles to primary amines was achieved by a homogeneous reduction with an excess of sodium borohydride and cobaltous chloride or with metals in liquid ammonia-methanol. Optimal results were obtained with the latter method; 83% of the substituents were converted into primary amines and 17% were lost by dealkylation.     

Identification of amino acids in the N-terminal domain of corticotropin-releasing factor receptor 1 that are important determinants of high-affinity ligand binding

The aim of the present study was to identify the N-terminal regions of human corticotropin-releasing factor (CRF) receptor type 1 (hCRF-R1) that are crucial for ligand binding. Mutant receptors were constructed by replacing specific residues in hCRF-R1 with amino acids from the corresponding position in the N-terminal region of the human vasoactive intestinal peptide receptor type 2 (hVIP-R2). In cyclic AMP stimulation and CRF binding assays, it was established that two regions within the N-terminal domain were crucial for the binding of CRF receptor agonists and antagonists: one region mapping to amino acids 43-50 and a second amino acid sequence extending from position 76 to 84 of hCRF-R1. Recently, it was found that the latter sequence plays a very important role in determining the high ligand selectivity of the Xenopus CRF-R1 (xCRF-R1). Replacement of amino acids 76-84 of hCRF-R1 with residues from the same segment of the hVIP-R2 N terminus markedly reduced the binding affinity of CRF ligands. Mutation of Arg76 or Asn81 but not Gly83 of hCRF-R1 to the corresponding amino acids of xCRF-R1 or hVIP-R2 resulted in 100-1,000-fold lower affinities for human/rat CRF, rat urocortin, and astressin. These data underline the importance of the N-terminal domain of CRF-R1 in high-affinity ligand binding.     

Identification of a novel contactin-associated transmembrane receptor with multiple domains implicated in protein-protein interactions

Receptor protein tyrosine phosphatase beta (RPTPbeta) expressed on the surface of glial cells binds to the glycosylphosphatidylinositol (GPI)-anchored recognition molecule contactin on neuronal cells leading to neurite outgrowth. We describe the cloning of a novel contactin-associated transmembrane receptor (p190/Caspr) containing a mosaic of domains implicated in protein-protein interactions. The extracellular domain of Caspr contains a neurophilin/coagulation factor homology domain, a region related to fibrinogen beta/gamma, epidermal growth factor-like repeats, neurexin motifs as well as unique PGY repeats found in a molluscan adhesive protein. The cytoplasmic domain of Caspr contains a proline-rich sequence capable of binding to a subclass of SH3 domains of signaling molecules. Caspr and contactin exist as a complex in rat brain and are bound to each other by means of lateral (cis) interactions in the plasma membrane. We propose that Caspr may function as a signaling component of contactin, enabling recruitment and activation of intracellular signaling pathways in neurons. The binding of RPTPbeta to the contactin-Caspr complex could provide a mechanism for cell-cell communication between glial cells and neurons during development.     

Expression and purification of the extracellular ligand binding region of metabotropic glutamate receptor subtype 1

Each metabotropic glutamate receptor possesses a large extracellular domain that consists of a sequence homologous to the bacterial periplasmic binding proteins and a cysteine-rich region. Previous experiments have proposed that the extracellular domain is responsible for ligand binding. However, it is currently unknown whether the extracellular ligand binding site can bind ligands without other domains of the receptor. We began by obtaining a sufficient amount of receptor protein on a baculovirus expression system. In addition to the transfer vector that encodes the entire coding region, transfer vectors that encode portions of the extracellular domain were designed. Here, we report a soluble metabotropic glutamate receptor that encodes only the extracellular domain and retains a ligand binding characteristic similar to that of the full-length receptor. The soluble receptor secreted into culture medium showed a dimerized form. Furthermore, we have succeeded in purifying it to homogeneity. Dose-response curves of agonists for the purified soluble receptor were examined. The effective concentration for half-maximal inhibition (IC50) of quisqualate for the soluble receptor was 3.8 x 10(-8) M, which was comparable to that for the full-length receptor. The rank order of inhibition of the agonists was quisqualate > ibotenate >/= L-glutamate approximately (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid. These data demonstrate that a ligand binding event in metabotropic glutamate receptors can be dissociated from the membrane domain.     

Cytoplasmic domains mediate the ligand-induced affinity shift of guanylyl cyclase C

Guanylyl cyclase C (GCC), the receptor for the Escherichia coli heat-stable enterotoxin (ST), exhibits multiple binding affinities, including high (RH) and low (RL) affinity sites and a ligand-induced conversion of low-affinity sites from a higher (RL1) to a lower (RL2) affinity state. Occupancy of the lowest affinity state of low-affinity sites is coupled to ligand-induced catalytic activation. In the present studies, ligand binding and catalytic activation properties of a series of intracellular deletion mutants of GCC were examined to identify the structural domains underlying expression of high- and low-affinity sites and the ligand-induced shift in low-affinity sites. These studies demonstrated that the cytoplasmic domains of GCC are not required, but extracellular and transmembrane domains are sufficient, for expression of high-affinity binding sites. In addition, the cytoplasmic juxtamembrane and kinase homology domains are required for expression of the ligand-induced affinity shift in low-affinity sites. Of significance, this shift in affinity was insensitive to adenine nucleotides, in contrast to other members of the receptor guanylyl cyclase family, such as guanylyl cyclase A (GCA). Also, the juxtamembrane and kinase homology domains are critical for coupling ST-receptor binding and guanylyl cyclase catalytic activation. Indeed, deletion of those domains from GCC results in a constitutively inhibited enzyme, suggesting that they function as positive effectors of ligand activation, in contrast to GCA and GCB, in which the kinase homology domain represses basal catalytic activity. These data suggest that the mechanisms regulating different members of the receptor guanylyl cyclase family are overlapping but not identical.     

Identification of an mRNA-binding protein and the specific elements that may mediate the pH-responsive induction of renal glutaminase mRNA

Various segments of the 3'-nontranslated region of the renal glutaminase (GA) mRNA were tested for their ability to enhance turnover and pH responsiveness. The combined effects were retained in the 340-base R-2 segment. However, the combined R-1 and R-3 fragments also imparted a partial destabilization and pH responsiveness to a chimeric beta-globin mRNA. RNA electrophoretic mobility shift assays indicated that cytosolic extracts of rat renal cortex contain a protein that binds to the R-2 and R-3 RNAs. The binding observed with the R-2 RNA was mapped to a direct repeat of an 8-base AU sequence. This binding was effectively competed with an excess of the same RNA, but not by adjacent or unrelated RNAs. UV cross-linking experiments identified a 48-kDa protein that binds to the AU repeats of the R-2 RNA. The apparent binding of this protein was greatly reduced in renal cytosolic extracts prepared from acutely acidotic rats. Two related RNA sequences in the R-3 segment also exhibited specific binding. However, the latter binding was more effectively competed by R-2 RNA than by itself, indicating that the homologous sites may be weaker binding sites for the same 48-kDa protein. Thus, a single protein may bind specifically to multiple instability elements within the 3'-nontranslated region of the GA mRNA and mediate its pH-responsive stabilization.     

Identification of amino acid residues that determine the differential ligand specificities of folate receptors alpha and beta

The homologous folate receptor (FR) types alpha and beta from both human and murine sources have opposite stereospecificities for reduced folate coenzymes and different affinities for a variety of (anti)folate compounds. The present study identifies the critical amino acid sequence divergence underlying functional differences between FR-alpha and FR-beta. Chimeric constructs of the cDNAs encoding human FR-alpha and FR-beta were expressed in human 293 fibroblasts. The resulting membrane associated proteins were characterized in terms of their ability to bind [3H]folic acid and their relative affinities for the (6S) and (6R) diastereoisomers of N5-methyltetrahydrofolate. Substitution of the amino-terminal portion (residues 1-92) in the mature FR-alpha polypeptide with the corresponding segment of FR-beta resulted in folate binding characteristics similar to FR-beta. Next, a series of chimeric constructs were generated, involving substitution of progressively shorter segments within residues 1-92 in FR-alpha with the corresponding peptides of FR-beta. In this fashion, it was determined that the alanine residue at position 49 in FR-alpha was critical for its functional divergence from FR-beta, since substitution at this position with Leu (the corresponding residue in FR-beta) resulted in the folate binding characteristics of FR-beta. Reciprocal substitution in FR-beta with peptide 1-92 of FR-alpha resulted in poor expression of a [3H]folic acid binding protein. By analysis of chimeric constructs, the poor [3H]folic acid binding of the FR-alpha(1-92)/beta(93-237) chimera could be attributed to interference of a short segment from FR-alpha in the vicinity of Ala 49 (peptide 39-59) with proper folding of the chimera. Conversion of the ligand binding properties of FR-beta to those of FR-alpha required the reciprocal mutation of Leu 49 to Ala, but in addition, substitution of one or more residues downstream of amino acid 92 of FR-beta with the corresponding residues in FR-alpha was essential. The homologous murine FR types alpha and beta, which are functionally analogous to the human receptor isoforms, also contain a similar Ala vs Leu substitution. These results indicate that steric/hydrophobic effects of the side chains of Leu vs Ala at position 49 will critically modulate the affinities and stereospecificities of FR isoforms for folate compounds. Furthermore, additional amino acid sequence divergence at one or more positions downstream of residue 92 in FR-alpha is also an essential determinant of the unique functional characteristics of this receptor isoform.     

Identification of functional domains of the human glutamate transporters EAAT1 and EAAT2

Glutamate transporters serve the important function of mediating removal of glutamate released at excitatory synapses and maintaining extracellular concentrations below excitotoxic levels. Excitatory amino acid transporter subtypes EAAT1 and EAAT2 have a high degree of sequence homology and similar predicted topology and yet display a number of functional differences. Several recombinant chimeric transporters were generated to identify domains that contribute to functional differences between EAAT1 and EAAT2. Wild-type transporters and chimeric transporters were expressed in Xenopus laevis oocytes, and electrogenic transport was studied under voltage clamp conditions. The differential sensitivity of EAAT1 and EAAT2 to transport blockers, kainate, threo-3-methylglutamate, and (2S, 4R)-4-methylglutamate as well as L-serine-O-sulfate transport and chloride permeability were employed to characterize chimeric transporters. One particular region, transmembrane domains 9 and 10, plays an important role in defining these functional differences. The intracellular carboxyl-terminal region may also play a minor role in conferring an effect on chloride permeability. This study provides important insight into the identification of functional domains that determine differences among glutamate transporter subtypes.     

Role of the extracellular domains of the cholecystokinin receptor in agonist binding

The cholecystokinin (CCK) receptor types A and B (CCKAR and CCKBR) are G protein-coupled receptors with approximately 50% amino acid identity; both have high affinity for the sulfated CCK octapeptide (CCK-8), whereas only the CCKBR has high affinity for gastrin. Previously, we identified five amino acids in the second extracellular loop (ECL) of the CCKBR that were essential for gastrin selectivity. Subsequent mutagenesis of one of these five amino acids (H207F) resulted in the loss of radiolabeled CCK-8 binding. CCK-8 stimulated total inositol phosphate accumulation in COS-1 cells transiently expressing the CCKBR-H207F with full efficacy and a 3044-fold reduced potency, which suggests that the loss of radioligand binding was caused by a loss in affinity. Alanine scanning mutagenesis was performed on the amino terminus near the top of transmembrane domain I (TMI) and on ECL1, two extracellular domains implicated in ligand binding by previous mutagenesis studies. 125I-Bolton-Hunter-CCK-8 binding to mutant receptors transiently expressed in COS-1 identified one nonconserved amino acid, R57A, at the top of TMI that caused a 21-fold reduction in CCK-8 affinity and four conserved amino acids, N115A, L116A, F120A and F122A, in the ECL1 that caused a 15.6-, 6-, 440-, and 8-fold reduction in affinity or efficacy. Alanine substitution of the equivalent amino acids in the CCKAR corresponding to each of the five amino acids in ECL1 and ECL2 affecting CCK-8 affinity for the CCKBR revealed only two mutations, L103A and F107A, that decreased CCK-8 affinity (68- and 2885-fold, respectively). These data suggest that CCK-8 interacts at multiple contact points in the extracellular domains of CCK receptors and that the CCKAR and CCKBR have distinct binding sites despite their shared high affinity for CCK-8.     

The CR1 and CR3 domains of the adenovirus type 5 E1A proteins can independently mediate activation of ATF-2

The adenovirus 12S E1A protein can stimulate the activity of the c-jun promoter through a conserved region 1 (CR1)-dependent mechanism. The effect is mediated by two AP-1/ATF-like elements, jun1 and jun2, that preferentially bind c-Jun-ATF-2 heterodimers. In this study, we show that the ATF-2 component of the c-Jun-ATF-2 heterodimer is the primary target for 12S E1A: 12S E1A can enhance the transactivating activity of the N terminus of ATF-2 when fused to a heterologous DNA-binding domain, whereas the transactivating activity of the c-Jun N terminus is not significantly affected. Activation of the ATF-2 N terminus by 12S E1A is dependent on CR1. In the context of the 13S E1A protein, CR1 and CR3 can both contribute to activation of ATF-2, and their relative contributions are dependent on the cell type. In contrast to activation of ATF-2 by stress-inducing agents, CR1-dependent activation of ATF-2 was found not to depend strictly on the presence of threonines 69 and 71 in the N terminus of ATF-2, which are targets for phosphorylation by stress-activated protein kinases (SAPKs). In agreement with this observation, we did not observe phosphorylation of threonines 69 and 71 or constitutively enhanced SAPK activity in E1A- plus E1B-transformed cell lines. These data suggest that CR1-dependent activation of ATF-2 by 12S E1A does not require phosphorylation of threonines 69 and 71 by SAPK.     

L-selectin binds to P-selectin glycoprotein ligand-1 on leukocytes: interactions between the lectin, epidermal growth factor, and consensus repeat domains of the selectins determine ligand binding specificity

The selectins mediate cellular interactions by binding carbohydrate determinants present on a limited number of glycoprotein ligands. L-selectin binds multiple ligands expressed on endothelial cells, while P-selectin interacts exclusively with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes. In this study, L-selectin was shown to bind leukocytes through the P-selectin ligand, PSGL-1, although at lower levels than P-selectin. L-selectin binding to PSGL-1 is specific since it was blocked by Abs to L-selectin or PSGL-1, required appropriate glycosylation of PSGL-1, and was Ca2+ dependent. The contributions of the extracellular domains of the selectins to ligand binding was assessed using a panel of chimeric selectins created by exchange of domains between L-selectin and P- or E-selectin. The lectin and epidermal growth factor domains of L- and P-selectin contributed significantly to binding through similar, if not identical, regions of PSGL-1. The different chimeric selectins revealed that the lectin domain was the dominant determinant for ligand binding, while cooperative interactions between the lectin, epidermal growth factor, and short consensus repeat domains of the selectins also modified ligand binding specificity. L-selectin binding to PSGL-1 expressed by leukocytes may mediate neutrophil rolling on stationary leukocytes bound to cytokine-induced endothelial cells, which was previously reported to be a L-selectin-dependent process.     

Identification of hyaluronan-binding domains of aggrecan

Aggrecan, a large cartilage proteoglycan, interacts with hyaluronan (HA), to form aggregates which function to resist compression in joints. The N-terminal region of aggrecan contains two structurally related globular domains, G1 and G2 separated by IGD domain. The G1 domain consists of three subdomains, A, B, and B', structural features characteristic to many other HA-binding proteoglycans. Here, we studied the interaction of aggrecan domains with HA using recombinant proteins expressed in 293 cells, an embryonal kidney cell line. Deglycosylation of the recombinant aggrecan fragment reduced the HA binding activity. We found that both the B and B' subdomains were required for HA binding and that a single module of A, B, or B' was unable to bind HA. The A subdomain increased the HA binding activity of the B-B' region. The G2 domain had no HA binding activity confirming previous reports. Studies of HA-binding properties using a BIAcoreTM biosensor system revealed that the KD of recombinant aggrecan fragment (AgW) consisting of G1, IGD, and G2 was 0.226 microM, whereas the KD of another HA-binding protein, native bovine link protein, is 0.089 microM. In contrast, AgMut11 which lacked subdomain A showed little HA binding activity. AgMut12 consisting of only B-B' had a 3.4-fold lower affinity and AgMut13 containing A-B-B' was 1.5-fold lower than AgW. These results suggest that carbohydrates are essential for high level aggrecan binding to HA and that the A subdomain of aggrecan functions in a cooperative manner with subdomains B and B'.     

Distinct domains of M-T2, the myxoma virus tumor necrosis factor (TNF) receptor homolog, mediate extracellular TNF binding and intracellular apoptosis inhibition

The myxoma virus tumor necrosis factor (TNF) receptor homolog, M-T2, is expressed both as a secreted glycoprotein that inhibits the cytolytic activity of rabbit TNF-alpha and as an endoglycosidase H-sensitive intracellular species that prevents myxoma virus-infected CD4+ T lymphocytes from undergoing apoptosis. To compare the domains of M-T2 mediating extracellular TNF inhibition and intracellular apoptosis inhibition, recombinant myxoma viruses expressing nested C-terminal truncations of M-T2 protein were constructed. One mutant, deltaL113, containing intact copies of only two cysteine-rich domains, was not secreted and was incapable of binding rabbit TNF-alpha yet retained full ability to inhibit virus-induced apoptosis of RL-5 cells. Thus, the minimal domain of intracellular M-T2 protein required to inhibit apoptosis is distinct from that required by the extracellular M-T2 for functional TNF-alpha binding and inhibition. This is the first report of a virus-encoded immunomodular protein with two distinct antiimmune properties.     

The pleckstrin homology and phosphotyrosine binding domains of insulin receptor substrate 1 mediate inhibition of apoptosis by insulin

Insulin and insulin-like growth factor 1 (IGF-1) evoke diverse biological effects through receptor-mediated tyrosine phosphorylation of insulin receptor substrate (IRS) proteins. We investigated the elements of IRS-1 signaling that inhibit apoptosis of interleukin 3 (IL-3)-deprived 32D myeloid progenitor cells. 32D cells have few insulin receptors and no IRS proteins; therefore, insulin failed to inhibit apoptosis during IL-3 withdrawal. Insulin stimulated mitogen-activated protein kinase in 32D cells expressing insulin receptors (32DIR) but failed to activate the phosphatidylinositol 3 (PI 3)-kinase cascade or to inhibit apoptosis. By contrast, insulin stimulated the PI 3-kinase cascade, inhibited apoptosis, and promoted replication of 32DIR cells expressing IRS-1. As expected, insulin did not stimulate PI 3-kinase in 32DIR cells, which expressed a truncated IRS-1 protein lacking the tail of tyrosine phosphorylation sites. However, this truncated IRS-1 protein, which retained the NH2-terminal pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains, mediated phosphorylation of PKB/akt, inhibition of apoptosis, and replication of 32DIR cells during insulin stimulation. These results suggest that a phosphotyrosine-independent mechanism mediated by the PH and PTB domains promoted antiapoptotic and growth actions of insulin. Although PI 3-kinase was not activated, its phospholipid products were required, since LY294002 inhibited these responses. Without IRS-1, a chimeric insulin receptor containing a tail of tyrosine phosphorylation sites derived from IRS-1 activated the PI 3-kinase cascade but failed to inhibit apoptosis. Thus, phosphotyrosine-independent IRS-1-linked pathways may be critical for survival and growth of IL-3-deprived 32D cells during insulin stimulation.