Structural insights into ligand recognition and selectivity of somatostatin receptors

Somatostatin receptors (SSTRs) play versatile roles in inhibiting the secretion of multiple hormones such as growth hormone and thyroid-stimulating hormone, and thus are considered as targets for treating multiple tumors. Despite great progress made in therapeutic development against this diverse receptor family, drugs that target SSTRs still show limited efficacy with preferential binding affinity and conspicuous side-effects. Here, we report five structures of SSTR2 and SSTR4 in different states, including two crystal structures of SSTR2 in complex with a selective peptide antagonist and a non-peptide agonist, respectively, a cryo-electron microscopy (cryo-EM) structure of G_i1-bound SSTR2 in the presence of the endogenous ligand SST-14, as well as two cryo-EM structures of G_i1-bound SSTR4 in complex with SST-14 and a small-molecule agonist J-2156, respectively. By comparison of the SSTR structures in different states, molecular mechanisms of agonism and antagonism were illustrated. Together with computational and functional analyses, the key determinants responsible for ligand recognition and selectivity of different SSTR subtypes and multiform binding modes of peptide and non-peptide ligands were identified. Insights gained in this study will help uncover ligand selectivity of various SSTRs and accelerate the development of new molecules with better efficacy by targeting SSTRs.Subject terms: Cryoelectron microscopy, X-ray crystallography     

Evolutionary history of the somatostatin and somatostatin receptors

Somatostatin and its receptors have a critical role in mammalian growth through their control pattern of secretion of growth hormone, but the evolutionary history of somatostatin and somatostatin receptors are ill defined. We used comparative whole genome analysis of Danio rerio, Carassius auratus, Xenopus tropicalis, Gallus gallus, Monodelphis domestica, Homo sapiens, Sus scrofa, Bos taurus, Mus musculus, Rattus norvegicus, Canis lupus familiaris, Ovis aries, Equus caballus, Pan troglodytes and Macaca mulatto to identify somatostatin and somatostatin receptors in each species. To date, we have identified a minimum of two genes of somatostatin and five somatostatin receptor genes in mammalian species with variable forms. We established a clear evolutionary history of the somatostatin system and traced the origin of the somatostatin system to 395 million years ago (MYA), identifying critical steps in their evolution.     

Characterization of functional receptors for somatostatin in rat pituitary cells in culture.

GH4C1 cells are a clonal strain of rat pituitary tumor cells which synthesize and secrete prolactin and growth hormone. Somatostatin, a hypothalamic tetradecapeptide, inhibits the release of growth hormone and, under certain circumstances, also prolactin from normal pituitary cells. We have prepared [125I-Tyr1]somatostatin (approximately 2200 C1/mmol) and have shown that this ligand binds to a limited number of high affinity sites on GH4C1 cells. Half-maximal binding of somatostatin occurred at a concentration of 6 x 10(-10) M. A maximum of 0.11 pmol of [125I-Tyr1]somatostatin was bound per mg of cell protein, equivalent to 13,000 receptor sites per cell. The rate constant for binding (kon) was 8 x 10(7) M(-1) min(-1). The rate constant for dissociation (koff) was determined by direct measurement to be 0.02 min(-1) both in the presence and absence of excess nonradioactive somatostatin. Binding of [125I-Tyr1]somatostatin was not inhibited by 10(-7) M thyrotropin-releasing hormones. Substance P, neurotensin, luteinizing hormone-releasing hormone, calcitonin, adrenocorticotropin, or insulin. Of seven nonpituitary cell lines tested, none had specific receptors for somatostatin. Somatostatin was shown to inhibit prolactin and growth hormone production by CH4C1 cells. The dose-response characteristics for binding and the biological actions of somatostatin were essentially coincident. Furthermore, among several clonal pituitary cell strains tested, only those which had receptors for somatostatin showed a biological response to the hormone. We conclude that the characterized somatostatin receptor is necessary for the biological actions of somatostatin on GH4C1 cells.     

Structural and functional neuroprotection in glaucoma: role of galantamine-mediated activation of muscarinic acetylcholine receptors

Glaucoma is the leading cause of irreversible blindness worldwide. Loss of vision due to glaucoma is caused by the selective death of retinal ganglion cells (RGCs). Treatments for glaucoma, limited to drugs or surgery to lower intraocular pressure (IOP), are insufficient. Therefore, a pressing medical need exists for more effective therapies to prevent vision loss in glaucoma patients. In this in vivo study, we demonstrate that systemic administration of galantamine, an acetylcholinesterase inhibitor, promotes protection of RGC soma and axons in a rat glaucoma model. Functional deficits caused by high IOP, assessed by recording visual evoked potentials from the superior colliculus, were improved by galantamine. These effects were not related to a reduction in IOP because galantamine did not change the pressure in glaucomatous eyes and it promoted neuronal survival after optic nerve axotomy, a pressure-independent model of RGC death. Importantly, we demonstrate that galantamine-induced ganglion cell survival occurred by activation of types M1 and M4 muscarinic acetylcholine receptors, while nicotinic receptors were not involved. These data provide the first evidence of the clinical potential of galantamine as neuroprotectant for glaucoma and other optic neuropathies, and identify muscarinic receptors as potential therapeutic targets for preventing vision loss in these blinding diseases.     

Structural analysis of the carbohydrate chains of beta-N-acetylhexosaminidases from bovine brain.

The oligosaccharide structures of bovine brain beta-N-acetylhexosaminidases A and B (EC 3.2.1.30) were studied at the glycopeptide level by employing 500 MHz 1H-n.m.r. spectroscopy and methylation analysis involving g.l.c.-m.s. More than 90% of the chains were found to be of the oligomannoside type, containing, on average, five to six mannose residues. Biantennary N-acetyl-lactosamine-type chains terminated in N-acetylneuraminic acid were found to comprise the remaining 5-10% of the total carbohydrate. The isoenzyme forms A and B do not differ from each other in the structure of their carbohydrate moiety, but do deviate in carbohydrate content and, in consequence, in the number of carbohydrate chains per molecule.     

Structural and functional studies of mammalian progesterone receptors

During the past years there has been an improvement in our understanding of the molecular mechanism of action of the progesterone receptor (PR). This was due to the obtention of monoclonal antibodies against PR which allowed the first structural analyses and led to the cloning of the genes.     

Structural studies of Fc receptors. III. Isolation and molecular weight analysis of the component chain of Fc gamma receptor of macrophage

Surface receptors of guinea pig peritoneal macrophages specific for the Fc region of IgG (Fc gamma receptor) were isolated and identified as a surface-radioiodinated component with a molecular weight of 44,000 that bound in an Fc-specific manner to IgG2 of guinea pig immunoglobulin immobilized in any of the following three different ways: IgG2 antibody in insoluble immune complex, IgG2 antibody bound to antigen-coupled Sepharose, and IgG2 covalently coupled to Sepharose. In order to obtain the Fc gamma receptor retaining the binding activity, the Fc-binding component was isolated by IgG2 affinity chromatography in which mild acidic buffer (pH 5.0-4.0) was chosen to elute the component bound to the affinity column. Forty-five to sixty-two percent of the eluted radioactivity was shown to rebind to the IgG2-affinity column. The bound fraction showed a single radioactive peak of 44,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Fc-binding component isolated by the affinity chromatography behaved similarly in gel filtration in the presence of a detergent, as did the detergent-solubilized Fc gamma receptor before isolation by affinity chromatography. These results suggested that the Fc gamma receptor was isolated in a native form. Furthermore, it was confirmed that the isolated Fc gamma receptor is distinct from actin or the actin-like protein (DNase I-binding protein) which had been reported to bind to IgG-affinity column.     

Critical role of peptidic toxins in the functional and structural analysis of nicotinic acetylcholine receptors

Animal toxins which interact on various receptors and channels have been often used in the studies of the functional roles of these targets. Nicotinic toxins have been purified from snake and cone venoms and are characterized by high affinity and various selectivity of interactions on the different nicotinic receptors subtypes. Since 30 years they have been used as molecular probes to identify, localize and purify these receptors. Furthermore, they have played a crucial role in the better understanding of their functional properties and have been useful in their structural studies. These peptidic toxins could be chemically synthetized or recombinantly expressed and nonnatural residues could be introduced in their sequences in order to delineate their functional interaction sites. The structural modelisation of toxin-nAChR interaction allows us to understand the antagonistic property of these toxins and open the way to the design of engineered ligands with predetermined specificity, useful as pharmacological tools or therapeutic agents in the numerous diseases involving this receptor family.     

Structural and functional characteristics of S1P receptors

The sphingosine-1-phosphate (S1P) family of G protein-coupled receptors (GPCR) regulates essential cellular processes such as proliferation, migration, cytoskeletal organization, adherens junction assembly, and morphogenesis. S1P, a product from the breakdown of sphingomyelin, binds to the five members of this receptor family, S1P(1), S1P(2), S1P(3), S1P(4), and S1P(5), previously referred to as endothelial differentiation gene (EDG)-1, -5, -3, -6, and -8. S1P receptors are widely expressed in different tissues, so it is not surprising that the S1P receptor family regulates many physiological processes, such as vascular maturation, cardiac development, lymphocyte trafficking, and vascular permeability. FTY720, a new S1P receptor agonist, is undergoing clinical trials as an immunosuppressor. Understanding the physiological role of these receptors and the basics of the ligand-receptor interaction will potentially provide new therapies to control a variety of diseases.     

Structural and functional characterization of the gamma 1 subunit of GABAA/benzodiazepine receptors.

The GABAA receptor gamma 1 subunit of human, rat and bovine origin was molecularly cloned and compared with the gamma 2 subunit in structure and function. Both gamma subunit variants share 74% sequence similarity and are prominently synthesized in often distinct areas of the central nervous system as documented by in situ hybridization. When co-expressed with alpha and beta subunits in Xenopus oocytes and mammalian cells, the gamma variants mediate the potentiation of GABA evoked currents by benzodiazepines and help generate high-affinity binding sites for these drugs. However, these sites show disparate pharmacological properties which, for receptors assembled from alpha 1, beta 1 and gamma 1 subunits, are characterized by the conspicuous loss in affinity for neutral antagonists (e.g. flumazenil) and negative modulators (e.g. DMCM). These findings reveal a pronounced effect of gamma subunit variants on GABAA/benzodiazepine receptor pharmacology.     

IgM and its receptors: Structural and functional aspects

This review combines the data obtained before the beginning of the 1990s with results published during the last two decades. The predominant form of the IgM molecule is a closed ring composed of five 7S subunits and a J chain. The new model of spatial structure of the pentamer postulates nonplanar mushroom-shaped form of the molecule with the plane formed by a radially-directed Fab regions and central protruding portion consisting of Cμ4 domains. Up to the year 2000 the only known Fc-receptor for IgM was pIgR. Interaction of IgM with pIgR results in secretory IgM formation, whose functions are poorly studied. The receptor designated as Fcα/μR is able to bind IgM and IgA. It is expressed on lymphocytes, follicular dendritic cells, and macrophages. A receptor binding IgM only named FcμR has also been described. It is expressed on T- and B-lymphocytes. The discovery of new Fc-receptors for IgM requires revision of notions that interactions between humoral reactions involving IgM and the cells of the immune system are mediated exclusively by complement receptors. In the whole organism, apart from IgM induced by immunization, natural antibodies (NA) are present and comprise in adults a considerable part of the circulating IgM. NA are polyreactive, germ-line-encoded, and emerge during embryogenesis without apparent antigenic stimuli. They demonstrate a broad spectrum of antibacterial activity and serve as first line of defense against microbial and viral infections. NA may be regarded as a transitional molecular form from invariable receptors of innate immunity to highly diverse receptors of adaptive immunity. By means of interaction with autoantigens, NA participate in maintenance of immunological tolerance and in clearance of dying cells. At the same time, NA may act as a pathogenic factor in atherosclerotic lesion formation and in development of tissue damage due to ischemia/reperfusion.     

Structural and functional understanding of the toll‐like receptors

Recognition of invading pathogens by the innate immune system is essential to initiate antimicrobial responses and trigger adaptive immunity. This is largely mediated by an array of pattern‐recognition receptor families that are essential for recognizing conserved molecular motifs characteristic of pathogenic microbes. One such family is the Toll‐like receptors (TLRs). Activation of TLRs induces production of pro‐inflammatory cytokines and type I interferons: the former triggers the synthesis of inflammatory mediators which cause fever, pain and other inflammation, and the latter mediates antiviral responses. Over the past decade, significant progress has been made in structural elucidation of TLRs in higher eukaryotes. The TLR structures with and without agonist and antagonist have been revealed by X‐ray crystallography and cryo‐electron microscopy studies, demonstrating the activated dimer formation induced by the agonistic ligand and the inhibition mechanism of the antagonistic ligand. Intracellular assembled structures and the TLR‐chaperone complex are also reported. As the structural understanding of TLRs becomes better integrated with biochemical and immunological studies, a more comprehensive picture of their architectural and functional properties will emerge. This review summarizes recent advances in structural biological and mechanistic studies on TLRs.     

Structural and functional aspects of the receptors for platelet-derived growth factor

Platelet-derived growth factor (PDGF) is a 30 kDa dimer of disulfide-bonded A and B chains. Three isoforms of PDGF have been isolated (PDGF-AA, PDGF-AB and PDGF-BB). These bind with different affinities and specificities to two structurally related cell surface receptors, viz. the α-receptor and the β-receptor. The receptors are transmembrane proteins with an intracellular, ligand-stimulatable protein tyrosine kinase domain. Activation of the receptors is intimately associated with receptor dimerization, and available data suggest that PDGF is a divalent ligand such that one molecule of PDGF binds and dimerizes two receptor molecules. Stimulation of PDGF receptors leads to a cascade of cellular events, which have been shown to require an intact receptor tyrosine kinase activity. However, ligand-induced internalization and degradation of the β-receptor occur essentially independent of the receptor kinase activity. Receptor activation leads to the phosphorylation on tyrosine residues of three enzymes, probably by direct phosphorylation: phospholipase C-γ, phosphatidylinositol 3′ kinase and Raf-1. In certain cells, PDGF β-receptor expression is inducible such that cells in normal tissue in vivo do not express receptors; only in inflammatory lesions or when cells are explanted in vitro, are receptors being expressed. Transformation by the v-sis oncogene is mediated by an autocrine PDGF-like growth factor. Although both the α- and β-receptors are structurally related to the v-fms and v-kit oncogenes, it is not known if the PDGF receptors have a transforming potential. In conclusion, the finding of three isoforms of PDGF that interact with two structurally related receptors implies a finely tuned regulatory network, the role of which in cell growth and transformation remains to be clarified.     

Structural and functional analysis of the complement component factor H with the use of different enzymes and monoclonal antibodies to factor H.

The action of six different enzymes on the function and structure of Factor H was investigated by use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, haemagglutination, two enzyme-linked immunosorbent assay systems and an assay for Factor I cofactor activity. Six monoclonal antibodies directed against the 38 kDa tryptic fragment of Factor H [which contains the binding site for C3b (a 180 kDa fragment of the third component of complement) and the cofactor activity] were also used to detect cleavage products derived from the same fragment. Elastase, chymotrypsin A4 or trypsin first cleaved Factor H to 36-38 kDa fragments carrying all six monoclonal anti-(Factor H)-binding sites. In parallel, the interaction of Factor H with surface-bound C3b was lost, whereas the cofactor function was preserved. Further cleavage of the 36-38 kDa fragments into two 13-19 kDa fragments (one carrying the MAH4 and MRC OX 24 epitopes, the other the MAH1, MAH2, MAH3 and MRC OX 23 epitopes) destroyed cofactor activity. Pepsin, bromelain or papain rapidly split off a 13-15 kDa fragment of Factor H carrying the MAH1, MAH2, MAH3 and MRC OX 23 epitopes and destroyed all tested functions of Factor H. Ficin cleaved Factor H into disulphide-linked fragments smaller than 25 kDa, but did not affect the functions of the Factor H molecule. The 38 kDa tryptic fragment of Factor H is the N-terminal end of the Factor H molecule, as determined by N-terminal sequence analysis. A model is presented of the substructure of Factor H.     

Tonic component of the conditioned reflex process and its functional role

Tonic background activity of 266 neurones in the hippocampus and different neocortical areas was studied in freely moving rabbits in the process of defensive and food instrumental conditioned performance and during switching-over of instrumental and classical food and defensive reflexes. Associations of CS and reinforcement evoke background activity changes in most of recorded cortical neurones preceding the development of other conditioned manifestations. Conditioned reflex was performed only after reaching the background firing rate of almost every examined neurone optimal for its realization. The performance of different conditioned reflexes was associated with different background activity levels of cortical neurones. The above mentioned data form the experimental basis for the identification of the tonic component in conditioned process which reflects tonic character of temporary connection formation and function.