Expression and structure of the human NGF receptor

The nucleotide sequence for the human nerve growth factor (NGF) receptor has been determined. The 3.8 kb receptor mRNA encodes a 427 amino acid protein containing a 28 amino acid signal peptide, an extracellular domain containing four 40 amino acid repeats with six cysteine residues at conserved positions followed by a serine/threonine-rich region, a single transmembrane domain, and a 155 amino acid cytoplasmic domain. The sequence of the extracellular domain of the NGF receptor predicts a highly ordered structure containing a negatively charged region that may serve as the ligand-binding site. This domain is conserved through evolution. Transfection of a full-length cDNA in mouse fibroblasts results in stable expression of NGF receptors that are recognized by monoclonal antibodies to the human NGF receptor and that bind [125I]NGF.     

Expression cloning of a receptor for murine granulocyte colony-stimulating factor

Two cDNAs encoding the receptor for murine granulocyte colony-stimulating factor (G-CSF) were isolated from a CDM8 expression library of mouse myeloid leukemia NFS-60 cells, and their nucleotide sequences were determined. Murine G-CSF receptor expressed in COS cells could bind G-CSF with an affinity and specificity similar to that of the native receptor expressed by mouse NFS-60 cells. The amino acid sequence encoded by the cDNAs has demonstrated that murine G-CSF receptor is an 812 amino acid polypeptide (Mr, 90,814) with a single transmembrane domain. The extracellular domain consists of 601 amino acids with a region of 220 amino acids that shows a remarkable similarity to rat prolactin receptor. The cytoplasmic domain of the G-CSF receptor shows a significant similarity with parts of the cytoplasmic domain of murine interleukin-4 receptor. A 3.7 kb mRNA coding for the G-CSF receptor could be detected in mouse myeloid leukemia NFS-60 and WEHI-3B D+ cells as well as in bone marrow cells.     

Molecular similarities in the ligand binding pockets of an odorant receptor and the metabotropic glutamate receptors

The 5.24 odorant receptor is an amino acid sensing receptor that is expressed in the olfactory epithelium of fish. The 5.24 receptor is a G-protein-coupled receptor that shares amino acid sequence identity to mammalian pheromone receptors, the calcium-sensing receptor, the T1R taste receptors, and the metabotropic glutamate receptors (mGluRs). It is most potently activated by the basic amino acids L-lysine and L-arginine. In this study we generated a homology model of the ligand binding domain of the 5.24 receptor based on the crystal structure of mGluR1 and examined the proposed lysine binding pocket using site-directed mutagenesis. Mutants of truncated glycosylated versions of the receptor containing only the extracellular domain were analyzed in a radioligand binding assay, whereas the analogous full-length membrane-bound mutants were studied using a fluorescence-based functional assay. In silico analysis predicted that aspartate 388 interacts with the terminal amino group on the side chain of the docked lysine molecule. This prediction was supported by experimental observations demonstrating that mutation of this residue caused a 26-fold reduction in the affinity for L-lysine but virtually no change in the affinity for the polar amino acid L-glutamine. In addition, mutations in four highly conserved residues (threonine 175, tyrosine 223, and aspartates 195 and 309) predicted to establish interactions with the alpha amino group of the bound lysine ligand greatly reduced or eliminated binding and receptor activation. These results define the essential features of amino acid selectivity within the 5.24 receptor binding pocket and highlight an evolutionarily conserved motif required for ligand recognition in amino acid activated receptors in the G-protein-coupled receptor superfamily.     

46 kd mannose 6-phosphate receptor: cloning, expression, and homology to the 215 kd mannose 6-phosphate receptor

We have isolated cDNA clones encoding the entire sequence of the bovine 46 kd cation-dependent mannose 6-phosphate (CD Man-6-P) receptor. Translation of CD Man-6-P receptor mRNA in Xenopus laevis oocytes results in a protein that binds specifically to phosphomannan-Sepharose, thus demonstrating that our cDNA clones encode a functional receptor. The deduced 279 amino acid sequence reveals a single polypeptide chain that contains a putative signal sequence and a transmembrane domain. Trypsin digestion of microsomal membranes containing the receptor and the location of the five potential N-linked glycosylation sites indicate that the receptor is a transmembrane protein with an extracytoplasmic amino terminus. This extracytoplasmic domain is homologous to the approximately 145 amino acid long repeating domains present in the 215 kd cation-independent Man-6-P receptor.     

Cloning, sequencing and expression of human TSH receptor

Complementary cDNA clones encoding the TSH (thyroid stimulatory hormone) receptor were isolated from a human thyroid lambda gt10 library using Iow stringency hybridization with LH/hCG (luteinizing hormone-human choriogonadotropic hormone) receptor probes. Sequencing of the clones showed a 764 amino acid open reading frame. The first 21 amino acids probably correspond to a signal peptide, the mature protein thus contains 743 amino acids (calculated molecular weight: 84,501 daltons). Its putative structure consists of a 394 amino acid extracellular domain, a 266 amino acid membrane spanning domain with 7 putative transmembrane segments and a 83 amino acid intracellular domain. A high degree of homology is observed with LH/hCG receptor suggesting the definition of a new subfamily of G-protein coupled receptors. Computer search showed the presence in the putative third intracellular loop of a motif resembling that described in the non receptor type protein tyrosine kinases (c-src, c-yes, c-fgr, etc...). RNA blots showed that the receptor messenger RNA consists of two major species of 4300 and 3900 nucleotides. The cDNA was inserted into an expression vector and after transfection into COS 7 cells it was shown to produce a functional TSH receptor.     

Efficient subgroup C avian sarcoma and leukosis virus receptor activity requires the IgV domain of the Tvc receptor and proper display on the cell membrane

We recently identified and cloned the receptor for subgroup C avian sarcoma and leukosis viruses [ASLV(C)], i.e., Tvc, a protein most closely related to mammalian butyrophilins, which are members of the immunoglobulin protein family. The extracellular domain of Tvc contains two immunoglobulin-like domains, IgV and IgC, which presumably each contain a disulfide bond important for native function of the protein. In this study, we have begun to identify the functional determinants of Tvc responsible for ASLV(C) receptor activity. We found that the IgV domain of the Tvc receptor is responsible for interacting with the glycoprotein of ASLV(C). Additional experiments demonstrated that a domain was necessary as a spacer between the IgV domain and the membrane-spanning domain for efficient Tvc receptor activity, most likely to orient the IgV domain a proper distance from the cell membrane. The effects on ASLV(C) glycoprotein binding and infection efficiency were also studied by site-directed mutagenesis of the cysteine residues of Tvc as well as conserved amino acid residues of the IgV Tvc domain compared to other IgV domains. In this initial analysis of Tvc determinants important for interacting with ASLV(C) glycoproteins, at least two aromatic amino acid residues in the IgV domain of Tvc, Trp-48 and Tyr-105, were identified as critical for efficient ASLV(C) infection. Interestingly, one or more aromatic amino acid residues have been identified as critical determinants in the other ASLV(A-E) receptors for a proper interaction with ASLV glycoproteins. This suggests that the ASLV glycoproteins may share a common mechanism of receptor interaction with an aromatic residue(s) on the receptor critical for triggering conformational changes in SU that initiate the fusion process required for efficient virus infection.     

The vitamin D receptor: a primitive steroid receptor related to thyroid hormone receptor

We have previously reported the cloning and sequencing of both the chicken and human vitamin D3 receptor cDNAs. A comparison of their deduced amino acid sequence with that of the other classic steroid hormone receptors and the receptor for thyroid hormone indicates that there are two regions of conservation between these molecules. The first is a 70 amino acid, cysteine-rich sequence (C1), the second region (C2) is a 62 amino acid region located towards the carboxyl terminus of the proteins. In other systems the former has been identified as a region responsible for DNA binding activity, whereas the latter represents the NH2-terminal boundary of the hormone binding domain. We present here evidence utilizing eucaryotic expression of cDNA encoding the hVDR C1 domain, followed by a DNA cellulose chromatography assay, which confirms that the DNA binding activity resides in this region of the receptor for vitamin D3. Additionally, the vitamin D3 receptor contains a 60 amino acid portion at its carboxyl terminus (C3) which exhibits homology with the receptor for thyroid hormone. Conservation in this region of the molecule is found only between homologous or closely related receptors. This indicates a relationship between the vitamin D3 receptor and the receptor for thyroid hormone and may suggest that they evolved from a single primordial gene.     

Molecular cloning and binding properties of the human type II activin receptor.

A full-length cDNA for the type II human activin receptor was cloned by hybridization from a human testis cDNA library. The sequence encodes a 513 amino acid protein that is 99% identical, at the amino acid level, with the mouse type II activin receptor. The type II human activin receptor consists of an extracellular domain that specifically binds activin A with a Kd of 360 pM, a single-membrane spanning domain, and an intracellular kinase domain with predicted serine/threonine specificity.     

A point mutation in the seventh hydrophobic domain of the alpha 2 adrenergic receptor increases its affinity for a family of beta receptor antagonists.

Previous studies have shown that differences in subtype-specific ligand binding between alpha 2 and beta 2 adrenergic receptors are largely determined by the seventh hydrophobic domain. Here, we report that a single amino acid substitution (Phe412----Asn) in the seventh hydrophobic domain of the alpha 2 adrenergic receptor reduces affinity for the alpha 2 antagonist yohimbine by 350-fold and increases affinity for beta antagonist alprenolol by 3000-fold. The affinity of this mutant receptor alpha 2F----N for several alpha and beta adrenergic receptor agonists and antagonists was determined. Beta adrenergic receptor antagonists containing an oxygen atom linking the amino side chain with the aromatic ring bound to alpha 2F----N with high affinity, while the beta receptor antagonist sotalol, which lacks this oxygen, bound with low affinity. These data suggest that the Asn residue is involved in conferring specificity for binding to a specific class of beta receptor antagonists.     

An interaction involving an arginine residue in the cytoplasmic domain of the 5-HT3A receptor contributes to receptor desensitization mechanism

A large cytoplasmic domain accounts for approximately one-third of the entire protein of one superfamily of ligand-gated membrane ion channels, which includes nicotinic acetylcholine (nACh), gamma-aminobutyric acid type A (GABA(A)), serotonin type 3 (5-HT3), and glycine receptors. Desensitization is one functional feature shared by these receptors. Because most molecular studies of receptor desensitization have focused on the agonist binding and channel pore domains, relatively little is known about the role of the large cytoplasmic domain (LCD) in this process. To address this issue, we sequentially deleted segments of the LCD of the 5-HT3A receptor and examined the function of the mutant receptors. Deletion of a small segment that contains three amino acid residues (425-427) significantly slowed the desensitization kinetics of the 5-HT3A receptor. Both deletion and point mutation of arginine 427 altered desensitization kinetics in a manner similar to that of the (425-427) deletion without significantly changing the apparent agonist affinity. The extent of receptor desensitization was positively correlated with the polarity of the amino acid residue at 427: the desensitization accelerates with increasing polarity. Whereas the R427L mutation produced the slowest desensitization, it did not significantly alter single channel conductance of 5-HT3A receptor. Thus, the arginine 427 residue in the LCD contributes to 5-HT3A receptor desensitization, possibly through forming an electrostatic interaction with its neighboring residues. Because the polarity of the amino acid residue at 427 is highly conserved, such a desensitization mechanism may occur in other members of the Cys-loop family of ligand-gated ion channels.     

Involvement of the conserved acidic amino acid domain of FGF receptor 1 in ligand-receptor interaction

The fibroblast growth factor receptor 1 (flg) contains eight acidic amino acids between the first and second immunoglobulin domain. This report examines the role of the acidic domain in the interaction of the flg receptor with its ligands. We observed a marked inhibition of binding of bFGF to the receptor when the acidic domain was completely deleted, but mutants with two and four amino acids deleted (flgΔ₂ and flgΔ₄, respectively) still bound the ligand. After addition of a bifunctional cross-linking reagent, cross-linked complexes (between bFGF and receptor) with the expected size were observed in cells expressing mutants lacking two or four acidic residues, but not in cells expressing mutants lacking six or eight acidic residues. Immunoprecipitation with anti-flg antibody followed by electrophoresis produced a band of 90 Kd in tunicamycin-treated cells expressing the mutant as well as the wild-type receptors, indicating that the inhibition of binding was not due to defective expression of the protein. The ability of flgΔ₈ to mediate a mitogenic response to FGFs was also greatly reduced when this mutated receptor was expressed in receptor-negative cells. The effect of replacing the acidic amino acids with lysine residues was also studied. Binding of bFGF to cells transfected with a plasmid encoding a mutated protein with four amino acid substitutions was totally inhibited, but an eight amino acid substitution did not alter ligand binding to the receptor. In this case the mutation with four amino acids substitution caused a drastic impairment of protein expression. Thus the acidic domain of the FGFR-1 plays an essential role in receptor function, either because it is important for a stable protein configuration or for ligand-receptor interaction. © 1993 Wiley-Liss, Inc.     

Antibodies to deduced sequences of the insulin receptor distinguish conserved and nonconserved regions in the IGF-I receptor

Antipeptide antibodies directed to two amino acid sequences predicted from the cDNA encoding the insulin proreceptor have been used to study the relationship between the human receptors for insulin and insulin-like growth factor I (IGF-I). An antibody directed to a cytoplasmic domain near the membrane spanning region of the proreceptor inhibited the protein tyrosine kinase activity of both receptors whereas an antibody directed to the C terminus of the insulin receptor showed no cross-reactivity with the IGF-I receptor. The results establish that the cloned cDNA from the human placenta encodes the insulin receptor and not the closely related IGF-I receptor, that the IGF-I and insulin receptors share a specific amino acid sequence necessary for the expression of enzymatic activity, and that the C terminus of the insulin receptor is not conserved in the IGF-I receptor.     

The Venus Fly Trap domain of the extracellular Ca2+ -sensing receptor is required for L-amino acid sensing

We previously demonstrated that the human calcium-sensing receptor (CaR) is allosterically activated by L-amino acids (Conigrave, A. D., Quinn, S. J., and Brown, E. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4814-4819). However, the domain-based location of amino acid binding has been uncertain. We now show that the Venus Fly Trap (VFT) domain of CaR, but none of its other major domains, is required for amino acid sensing. Several constructs were informative when expressed in HEK293 cells. First, the wild-type CaR exhibited allosteric activation by L-amino acids as previously observed. Second, two CaR-mGlu chimeric receptor constructs that retained the VFT domain of CaR, one containing the extracellular Cys-rich region of CaR and the other containing the Cys-rich region of the rat metabotropic glutamate type-1 (mGlu-1) receptor, together with the rat mGlu-1 transmembrane region and C-terminal tail, retained amino acid sensing. Third, a CaR lacking residues 1-599 of the N-terminal extracellular head but retaining an intact CaR transmembrane region and a functional but truncated C terminus (headless-T903 CaR) failed to respond to L-amino acids but retained responsiveness to the type-II calcimimetic NPS R-467. Finally, a T903 CaR control that retained an intact N terminus also retained L-amino acid sensing. Taken together, the data indicate that the VFT domain of CaR is necessary for L-amino acid sensing and are consistent with the hypothesis that the VFT domain is the site of L-amino acid binding. The findings support the concept that the mGlu-1 amino acid binding site for L-glutamate is conserved as an L-amino acid binding site in its homolog, the CaR.     

Proline residue 280 in the second extracellular loop (EC2) of the VPAC2 receptor is essential for the receptor structure

Inspection of the amino acid sequence of the human VPAC1 and the VPAC2 receptors after alignment of the conserved residues indicates that the second extracellular loop (EC2) is one amino acid shorter in the VPAC1 receptor due to the lack of a proline residue in position 294. We hypothesized that this could be of importance for receptor structure and/or for ligand recognition. Insertion by directed mutagenesis of a proline in that position (294 VPAC1) had little consequence on the binding of several agonists but reduced the affinity for the VPAC1 antagonist. Coupling of the 294 VPAC1 receptor to adenylate cyclase was improved, as demonstrated by an increased affinity for VIP and other agonists, and by a shift of the VPAC1 antagonist to partial agonist behavior. Deletion of the proline 280 (DeltaPro280 VPAC2) in the VPAC2 receptor markedly reduced the apparent affinity for all the agonists tested. Replacement of the proline by a glycine residue had a smaller effect on the ligands affinities. The proline residue in the VPAC2 receptor EC2 is thus essential for the receptor structure, and the EC2 domain is involved in ligand recognition and receptor functionality.     

Mutational Analysis Defines a C-Terminal Tail Domain of Rap1 Essential for Telomeric Silencing in Saccharomyces Cerevisiae

Alleles specifically defective in telomeric silencing were generated by in vitro mutagenesis of the yeast RAP1 gene. The most severe phenotypes occur with three mutations in the C-terminal 28 amino acids. Two of the alleles are nonsense mutations resulting in truncated repressor/activator protein 1 (RAP1) species lacking the C-terminal 25-28 amino acids; the third allele is a missense mutation within this region. These alleles define a novel 28-amino acid region, termed the C-terminal tail domain, that is essential for telomeric and HML silencing. Using site-directed mutagenesis, an 8-amino acid region (amino acids 818-825) that is essential for telomeric silencing has been localized within this domain. Further characterization of these alleles has indicated that the C-terminal tail domain also plays a role in telomere size control. The function of the C-terminal tail in telomere maintenance is not mediated through the RAP1 interacting factor RIF1: rap1 alleles defective in both the C-terminal tail and RIF1 interaction domains have additive effects on telomere length. Overproduction of SIR3, a dose-dependent enhancer of telomeric silencing, suppresses the telomeric silencing, but not length, phenotypes of a subset of C-terminal tail alleles. In contrast, an allele that truncates the terminal 28 amino acids of RAP1 is refractory to SIR3 overproduction. These results indicate that the C-terminal tail domain is required for SIR3-dependent enhancement of telomeric silencing. These data also suggest a distinct set of C-terminal requirements for telomere size control and telomeric silencing.     

Expression cloning and characterization of the TGF-beta type III receptor.

The rat TGF-beta type III receptor cDNA has been cloned by overexpression in COS cells. The encoded receptor is an 853 amino acid protein with a large N-terminal extracellular domain containing at least one site for glycosaminoglycan addition, a single hydrophobic transmembrane domain, and a 41 amino acid cytoplasmic tail with no obvious signaling motif. Introduction of the cDNA into COS cells and L6 myoblasts induces expression of a heterogenously glycosylated 280-330 kd protein characteristic of the type III receptor that binds TGF-beta 1 specifically. In L6 myoblasts lacking the endogenous type III receptor, expression of the recombinant receptor leads to an increase in the amount of ligand bound and cross-linked to surface type II TGF-beta receptors. This indicates that the type III receptor may regulate the ligand-binding ability or surface expression of the type II receptor.