Identification of ligand binding determinants of the prolactin receptor.

The prolactin (PRL) receptor, a lactogen- and primate somatogen-binding protein, is a member of an expanding superfamily (cytokine/growth hormone (GH)/PRL) of single membrane-spanning receptors. Two features commonly shared among this group of proteins are the presence of two pairs of cysteines, generally found in the N-terminal region of the extracellular domain, and a WSxWS (WS) motif, frequently located proximal to the transmembrane domain. We have recently shown the 4 cysteines to be critical to the maintenance of the structural and functional integrity of the PRL-receptor. In the present study, we prepared a set of eight chimeric rat PRL/human GH receptors and several alanine mutants, to assess the importance of the Cys-rich domain (residues 12-68) in confering specificity to PRL binding. The role of the WS motif in high affinity binding was also investigated. Binding of 125I-labeled ovine PRL or human GH to membrane preparations from COS-7 cells transiently expressing the mutant receptors have defined a region within the first disulfide loop (residues Arg13, Asp16, Glu18) and the set of lactogen-specific sequences between the two pairs of cysteines as key determinants of PRL-binding specificity, which converge to form a patch on a two-dimensional model of the PRL receptor. We also demonstrate that, although PRL- and GH-specific determinants overlap in certain areas, they are not identical. Finally, substitution of the WS motif with alanine residues precludes high affinity binding to ovine PRL and human GH and suggests that this structural element may provide a target site for the interaction of an accessory protein necessary for the formation of a high-affinity receptor complex.     

Structure and ligand specificity of the Drosophila melanogaster insulin receptor.

The insulin-binding and protein tyrosine kinase subunits of the Drosophila melanogaster insulin receptor homolog have been identified and characterized by using antipeptide antibodies elicited to the deduced amino acid sequence of the alpha and beta subunits of the human insulin receptor. In D. melanogaster embryos and cell lines, the insulin receptor contains insulin-binding alpha subunits of 110 or 120 kilodaltons (kDa), a 95-kDa beta subunit that is phosphorylated on tyrosine in response to insulin in intact cells and in vitro, and a 170-kDa protein that may be an incompletely processed receptor. All of the components are synthesized from a proreceptor, joined by disulfide bonds, and exposed on the cell surface. The beta subunit is recognized by an antipeptide antibody elicited to amino acids 1142 to 1162 of the human insulin proreceptor, and the alpha subunit is recognized by an antipeptide antibody elicited to amino acids 702 to 723 of the human proreceptor. Of the polypeptide ligands tested, only insulin reacts with the D. melanogaster receptor. Insulinlike growth factors type I and II, epidermal growth factor, and the silkworm insulinlike prothoracicotropic hormone are unable to stimulate autophosphorylation. Thus despite the evolutionary divergence of vertebrates and invertebrates, the essential features of the structure and intrinsic functions of the insulin receptor have been remarkably conserved.     

Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity.

To identify structural characteristics of the closely related cell surface receptors for insulin and IGF-I that define their distinct physiological roles, we determined the complete primary structure of the human IGF-I receptor from cloned cDNA. The deduced sequence predicts a 1367 amino acid receptor precursor, including a 30-residue signal peptide, which is removed during translocation of the nascent polypeptide chain. The 1337 residue, unmodified proreceptor polypeptide has a predicted Mr of 151,869, which compares with the 180,000 Mr IGF-I receptor precursor. In analogy with the 152,784 Mr insulin receptor precursor, cleavage of the Arg-Lys-Arg-Arg sequence at position 707 of the IGF-I receptor precursor will generate alpha (80,423 Mr) and beta (70,866 Mr) subunits, which compare with approximately 135,000 Mr (alpha) and 90,000 Mr (beta) fully glycosylated subunits.     

Changing the insulin receptor to possess insulin-like growth factor I ligand specificity

To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, we prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the alpha-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor.     

The insulin receptor-related receptor. Tissue expression, ligand binding specificity, and signaling capabilities.

In 1989, Shier and Watt identified a gene which was predicted to encode a new member of the insulin receptor (IR) family, and they called it the insulin receptor-related receptor (IRR) (Shier, P., and Watt, V. M. (1989) J. Biol. Chem. 264, 14605-14608). However, the tissues expressing this receptor, its ligand binding specificity and its signaling capability have remained unknown. In the present studies we report Northern blot analyses and polymerase chain reaction data, which indicate that the IRR mRNA is expressed in a variety of tissues, including the human kidney, heart, skeletal muscle, liver, and pancreas. In order to examine the ligand(s) recognized by IRR, we constructed a chimeric receptor with the extracellular domain of the IR replaced with that of IRR. This chimera was found not to bind radioactively labeled insulin, insulin-like growth factor I (IGF-I), or IGF-II. These ligands and relaxin, the only other known member of the mammalian insulin family, also failed to stimulate the tyrosine kinase activity of this chimeric receptor. A second chimeric receptor with the extracellular domain of IR and the kinase domain of IRR was also constructed and utilized to study the signaling capabilities of the kinase domain of IRR. This chimera exhibited high affinity insulin binding and insulin-stimulated tyrosine kinase activity. The kinase domains of the IR and IRR were found capable of phosphorylating the same spectrum of exogenous and endogenous substrates. However, Chinese hamster ovary (CHO) cells stably overexpressing the kinase domain of IRR exhibited elevated basal thymidine incorporation and 2-deoxyglucose uptake compared with CHO cells and CHO cells overexpressing wild-type IR. We conclude that: 1) IRR is expressed in the human kidney, heart, skeletal muscle, liver, and pancreas, 2) IRR does not appear to be the receptor of any known member of the insulin family, and 3) the tyrosine kinase of IRR appears to be similar to that of IR in both the spectrum of substrates phosphorylated and the biological responses stimulated.     

Spatial determinants of specificity in insulin action

Insulin is a potent stimulator of intermediary metabolism, however the basis for the remarkable specificity of insulin's stimulation of these pathways remains largely unknown. This review focuses on the role compartmentalization plays in insulin action, both in signal initiation and in signal reception. Two examples are discussed: (1) a novel signalling pathway leading to the phosphorylation of the caveolar coat protein caveolin, and (2) a recently identified scaffolding protein, PTG, involved directly in the regulation of enzymes controlling glycogen metabolism.     

Identification of XcpP domains that confer functionality and specificity to the Pseudomonas aeruginosa type II secretion apparatus

Gram-negative bacteria have evolved several types of secretion mechanisms to release proteins into the extracellular medium. One such mechanism, the type II secretory system, is a widely conserved two-step process. The first step is the translocation of signal peptide-bearing exoproteins across the inner membrane. The second step, the translocation across the outer membrane, involves the type II secretory apparatus or secreton. The secretons are made up of 12-15 proteins (Gsp) depending on the organism. Even though the systems are conserved, heterologous secretion is mostly species restricted. Moreover, components of the secreton are not systematically exchangeable, especially with distantly related microorganisms. In closely related species, two components, the GspC and GspD (secretin) family members, confer specificity for substrate recognition and/or secreton assembly. We used Pseudomonas aeruginosa as a model organism to determine which domains of XcpP (GspC member) are involved in specificity. By constructing hybrids between XcpP and OutC, the Erwinia chrysanthemi homologue, we identified a region of 35 residues that was not exchangeable. We showed that this region might influence the stability of the XcpYZ secreton subcomplex. Remarkably, XcpP and OutC have domains, coiled-coil and PDZ, respectively, which exhibit the same function but that are structurally different. Those two domains are exchangeable and we provided evidence that they are involved in the formation of homomultimeric complexes of XcpP.     

Discovery of phage determinants that confer sensitivity to bacterial immune systems

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Identification of the domain in the human interleukin-11 receptor that mediates ligand binding

The interleukin-11 receptor (IL-11R) belongs to the hematopoietic receptor superfamily. The functional receptor complex comprises IL-11, IL-11R and the signal-transducing subunit gp130. The extracellular part of the IL-11R consists of three domains: an N-terminal immunoglobulin-like domain, D1, and two fibronectin-type III-like (FNIII) domains and D2 and D3. The two FNIII domains comprise the cytokine receptor-homology region defined by a set of four conserved cysteine residues in the N-terminal domain (D2) and a WSXWS sequence motif in the C-terminal domain (D3). We investigated the structural and functional role of the third extracellular receptor domain of IL-11R. A molecular model of the human IL-11/IL-11R complex allowed the identification of amino acid residues in IL-11R to be involved in ligand binding. Most of them were located in the third extracellular domain, which therefore should be able to bind with high affinity to IL-11. To prove this prediction, domain D3 of the IL-11R was expressed in Escherichia coli, refolded and purified. For structural characterization, circular dichroism, fluorescence and NMR spectroscopy were used. By plasmon resonance experiments, we show that the ligand-binding capacity of this domain is as high as that one for the whole receptor. These results provide a basis for further structural investigations that could be used for the rational design of potential agonists and antagonists essential in human therapy.     

A chicken gonadotropin-releasing hormone receptor that confers agonist activity to mammalian antagonists. Identification of D-Lys(6) in the ligand and extracellular loop two of the receptor as determinants

Mammalian receptors for gonadotropin-releasing hormone (GnRH) have over 85% sequence homology and similar ligand selectivity. Biological studies indicated that the chicken GnRH receptor has a distinct pharmacology, and certain antagonists of mammalian GnRH receptors function as agonists. To explore the structural determinants of this, we have cloned a chicken pituitary GnRH receptor and demonstrated that it has marked differences in primary amino acid sequence (59% homology) and in its interactions with GnRH analogs. The chicken GnRH receptor had high affinity for mammalian GnRH (K(i) 4.1 +/- 1.2 nM), similar to the human receptor (K(i) 4.8 +/- 1.2 nM). But, in contrast to the human receptor, it also had high affinity for chicken GnRH ([Gln(8)]GnRH) and GnRH II ([His(5),Trp(7),Tyr(8)]GnRH) (K(i) 5.3 +/- 0.5 and 0.6 +/- 0.01 nM). Three mammalian receptor antagonists were also pure antagonists in the chicken GnRH receptor. Another three, characterized by D-Lys(6) or D-isopropyl-Lys(6) moieties, functioned as pure antagonists in the human receptor but were full or partial agonists in the chicken receptor. This suggests that the Lys side chain interacts with functional groups of the chicken GnRH receptor to stabilize it in the active conformation and that these groups are not available in the activated human GnRH receptor. Substitution of the human receptor extracellular loop two with the chicken extracellular loop two identified this domain as capable of conferring agonist activity to mammalian antagonists. Although functioning of antagonists as agonists has been shown to be species-dependent for several GPCRs, the dependence of this on an extracellular domain has not been described.     

The ligand specificity of the G-protein-coupled receptor GPR34

Lyso-PS (lyso-phosphatidylserine) has been shown to activate the G(i/o)-protein-coupled receptor GPR34. Since in vitro and in vivo studies provided controversial results in assigning lyso-PS as the endogenous agonist for GPR34, we investigated the evolutionary conservation of agonist specificity in more detail. Except for some fish GPR34 subtypes, lyso-PS has no or very weak agonistic activity at most vertebrate GPR34 orthologues investigated. Using chimaeras we identified single positions in the second extracellular loop and the transmembrane helix 5 of carp subtype 2a that, if transferred to the human orthologue, enabled lyso-PS to activate the human GPR34. Significant improvement of agonist efficacy by changing only a few positions strongly argues against the hypothesis that nature optimized GPR34 as the receptor for lyso-PS. Phylogenetic analysis revealed several positions in some fish GPR34 orthologues which are under positive selection. These structural changes may indicate functional specification of these orthologues which can explain the species- and subtype-specific pharmacology of lyso-PS. Furthermore, we identified aminoethyl-carbamoyl ATP as an antagonist of carp GPR34, indicating ligand promiscuity with non-lipid compounds. The results of the present study suggest that lyso-PS has only a random agonistic activity at some GPR34 orthologues and the search for the endogenous agonist should consider additional chemical entities.     

Identification of determinants of ligand binding affinity and selectivity in the prostaglandin D2 receptor CRTH2

The chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is a G protein-coupled receptor that mediates the pro-inflammatory effects of prostaglandin D(2) (PGD(2)) generated in allergic inflammation. The CRTH2 receptor shares greatest sequence similarity with chemoattractant receptors compared with prostanoid receptors. To investigate the structural determinants of CRTH2 ligand binding, we performed site-directed mutagenesis of putative mCRTH2 ligand-binding residues, and we evaluated mutant receptor ligand binding and functional properties. Substitution of alanine at each of three residues in the transmembrane (TM) helical domains (His-106, TM III; Lys-209, TM V; and Glu-268, TM VI) and one in extracellular loop II (Arg-178) decreased PGD(2) binding affinity, suggesting that these residues play a role in binding PGD(2). In contrast, the H106A and E268A mutants bound indomethacin, a nonsteroidal anti-inflammatory drug, with an affinity similar to the wild-type receptor. HEK293 cells expressing the H106A, K209A, and E268A mutants displayed reduced inhibition of intracellular cAMP and chemotaxis in response to PGD(2), whereas the H106A and E268A mutants had functional responses to indomethacin similar to the wild-type receptor. Binding of PGE(2) by the E268A mutant was enhanced compared with the wild-type receptor, suggesting that Glu-268 plays a role in determining prostanoid ligand selectivity. Replacement of Tyr-261 with phenylalanine did not affect PGD(2) binding but decreased the binding affinity for indomethacin. These results provided the first details of the ligand binding pocket of an eicosanoid-binding chemoattractant receptor.     

Structural determinants of natriuretic peptide receptor specificity and degeneracy

Cardiovascular homeostasis and blood pressure regulation are reliant, in part, on interactions between natriuretic peptide (NP) hormones and natriuretic peptide receptors (NPR). The C-type NPR (NPR-C) is responsible for clearance of NP hormones from the circulation, and displays a cross-reactivity for all NP hormones (ANP, BNP, and CNP), in contrast to other NPRs, which are more restricted in their specificity. In order to elucidate the structural determinants for the binding specificity and cross-reactivity of NPR-C with NP hormones, we have determined the crystal structures of the complexes of NPR-C with atrial natriuretic peptide (ANP), and with brain natriuretic peptide (BNP). A structural comparison of these complexes, with the previous structure of the NPR-C/CNP complex, reveals that NPR-C uses a conformationally inflexible surface to bind three different, highly flexible, NP ligands. The complex structures support a mechanism of rigid promiscuity rather than conformational plasticity by the receptor. While ANP and BNP appear to adopt similar receptor-bound conformations, the CNP structure diverges, yet shares sets of common receptor contacts with the other ligands. The degenerate versus selective hormone recognition properties of different NPRs appears to derive largely from two cavities on the receptor surfaces, pocket I and pocket II, that serve as anchoring sites for hormone side-chains and modulate receptor selectivity.     

Identification of tyrosine phosphatases that dephosphorylate the insulin receptor. A brute force approach based on "substrate-trapping" mutants

Many pharmacologically important receptors, including all cytokine receptors, signal via tyrosine (auto)phosphorylation, followed by resetting to their original state through the action of protein tyrosine phosphatases (PTPs). Establishing the specificity of PTPs for receptor substrates is critical both for understanding how signaling is regulated and for the development of specific PTP inhibitors that act as ligand mimetics. We have set up a systematic approach for finding PTPs that are specific for a receptor and have validated this approach with the insulin receptor kinase. We have tested nearly all known human PTPs (45) in a membrane binding assay, using "substrate-trapping" PTP mutants. These results, combined with secondary dephosphorylation tests, confirm and extend earlier findings that PTP-1b and T-cell PTP are physiological enzymes for the insulin receptor kinase. We demonstrate that this approach can rapidly reduce the number of PTPs that have a particular receptor or other phosphoprotein as their substrate.     

Characterization and ligand specificity of sheep IgG2 receptor

Neutrophils and macrophages in cattle express a novel class of immunoglobulin Fc receptor, specific for bovine IgG2, termed boFcγ2R. In cows, the ability of neutrophils to kill immunoglobulin-opsonized microorganisms appears to depend largely on this subclass. Although related to other mammalian FcγRs, boFcγ2R belongs to a novel gene family that includes the human killer Ig-like receptor and FcαRI (CD89) proteins. In this study, we describe the presence and characterization of this novel class of FcγR in sheep. The comparative analysis of this novel FcγR has allowed us to begin an exploration of some immunological characteristic of ruminants. The GenBank accession number of the nucleotide sequence reported here is EF541479 and FJ198054.     

Identification of two determinants that attenuate vaccine-related type 2 poliovirus.

The poliovirus P2/P712 strain is an attenuated virus that is closely related to the type 2 Sabin vaccine strain. By using a mouse model for poliomyelitis, sequences responsible for attenuation of the P2/P712 strain were previously mapped to the 5' noncoding region of the genome and a central region encoding VP1, 2Apro, 2B, and part of 2C. To identify specific determinants that attenuate the P2/P712 strain, recombinants between this virus and the mouse-adapted P2/Lansing were constructed and their neurovirulence in mice was determined. By using this approach, the attenuation determinant in the central region was mapped to capsid protein VP1. Candidate attenuating sequences in VP1 and the 5' noncoding region were identified by comparing the P2/P712 sequence with that of vaccine-associated isolate P2/P117, and the P2/117 sequences were introduced into the P2/Lansing-P2/P712 recombinants by site-directed mutagenesis. Results of neurovirulence assays in mice indicate that an A at nucleotide 481 in the 5' noncoding region and isoleucine (Ile) at position 143 of capsid protein VP1 are the major determinants of attenuation of P2/P712. These determinants also attenuated neurovirulence in transgenic mice expressing human poliovirus receptors, a new model for poliomyelitis in which virulent viruses are not host restricted. These results demonstrate that A-481 and Ile-143 are general determinants of attenuation.