Selective regulation of somatostatin receptor subtype signaling: evidence for constitutive receptor activation

Anterior pituitary hormone secretion is under tonic suppression by hypothalamic somatostatin signaling through somatostatin receptor subtypes (SSTs). Because some hormonal axes are known to be abnormally regulated by ligand-independent constitutively active G protein-coupled receptors, we tested pituitary SSTs for selective constitutive signaling. We therefore differentially silenced endogenous SST2, SST3, and SST5 in somatostatin-sensitive ACTH-secreting mouse AtT-20 pituitary corticotroph cells using small inhibitory RNA (siRNA) and analyzed downstream SSTs-regulated pathways. Transfection with siRNA reduced specific receptor subtype mRNA expression up to 82%. Specificity of receptor silencing was validated against negative controls with different gene-selective siRNAs, concordance of mRNA and cAMP changes, reduced potency of receptor-selective agonists, and phenotype rescue by overexpression of the silenced receptor. Mouse SST3 > SST5 > SST2 knockdown increased basal cAMP accumulation (up to 200%) and ACTH secretion (up to 60%). SST2- and SST5-selective agonist potencies were reduced by SST3- and SST5-silencing, respectively. SST5 > SST2 = SST3 silencing also increased basal levels of ERK1/2 phosphorylation. SST3- and SST5-knockdown increased cAMP was only partially blocked by pertussis toxin. The results show that SST2, SST3, and SST5 exhibit constitutive activity in mouse pituitary corticotroph cells, restraining adenylate cyclase and MAPK activation and ACTH secretion. SST3 mainly inhibits cAMP accumulation and ACTH secretion, whereas SST5 predominantly suppresses MAPK pathway activation. Therefore, SST receptor subtypes control pituitary cell function not only through somatostatin binding to variably expressed cell membrane receptor subtypes, but also by differential ligand-independent receptor-selective constitutive action.     

Characterization of somatostatin receptor subtype 2 expression in stably transfected A-427 human cancer cells

Although radiolabeled somatostatin analogs have become highly prevalent in the diagnosis and treatment of somatostatin receptor subtype (sst)-positive tumors, there are relatively few options with respect to sst-positive tumor cell lines and animal models. It would be highly beneficial, particularly for therapeutic purposes, to have several clones of one human sst2-positive cell line that express a range of sst2 concentrations for evaluating the dose response and intracellular processing of radiolabeled somatostatin analogs. The human non-small cell lung cancer line A-427 was stably transfected with a hemagglutinin-tagged human sst2. Expression of the receptor was evaluated in vitro using flow cytometry, saturation binding analysis, internalization assays, and quantitative polymerase chain reaction. The receptor expression was also validated in an in vivo mouse model in biodistribution and micro-positron emission tomography (microPET) studies using the somatostatin analog octreotide (OC), which was linked to the (64)Cu chelator 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), or (64)Cu-TETA-OC. Stable clones were isolated, and four clones (2, 4, 5, and 7) were chosen for further examination. In vitro assays showed that clone 4 had no expression of sst2, whereas the others had various levels in the order of 7 > 2 > 5. Biodistribution studies with (64)Cu-TETA-OC showed the same rank order, with tumor uptake of the clones ranging from 0.8 to 6.5% injected dose/g. These studies showed that there was a strong correlation among the in vitro assays and between the in vitro assays and the biodistribution. MicroPET confirmed significant uptake of (64)Cu-TETA-OC in clone 7 and background uptake in clone 4. These studies show that clones of a human cell line can be produced expressing various levels of sst2 that should be useful for the future evaluation of radiolabeled somatostatin analogs.     

多药耐药基因的转录调控与治疗学机会

人肿瘤多药耐药mdr-1基因的转录调控机制复杂。多个正调控和负调控元件与转录因子的相互作用、表遗传学因素的参与等共同决定着人mdr-1基因表达的组织、细胞和刺激的特异性和依赖性。同时,也为特异或相对特异性地防止／抑制肿瘤细胞的mdr-1基因表达、克服肿瘤多药耐药性提供了基础。新抗肿瘤药物沙尔威辛和ET-743有力地诠释了控制mdr-1基因转录所蕴藏的治疗学机会。     

Identification and characterization of subtype selective somatostatin receptor agonists.

High affinity, subtype selective non-peptide agonists of somatostatin receptor subtypes 1-5 were identified in combinatorial libraries constructed based on molecular modeling of known peptide agonists. Simultaneous traditional chemical synthesis yielded an additional series of somatostatin subtype-2 receptor (SSTR2) selective agonists. These compounds have been used to further define the physiological functions of the individual somatostatin receptor subtypes. In vitro experiments demonstrated the role of the SSTR2 in inhibition of glucagon release from mouse pancreatic alpha-cells and the somatostatin subtype-5 receptor (SSTR5) as a mediator of insulin secretion from pancreatic beta-cells. Both SSTR2 and SSTR5 regulated growth hormone release from the rat anterior pituitary gland. In vivo studies performed with SSTR2 receptor selective compounds demonstrated effective inhibition of pulsatile growth hormone release in rats. The SSTR2 selective compounds also lowered plasma glucose levels in normal and diabetic animal models. The availability of high affinity, subtype selective non-peptide agonists for each of the somatostatin receptors provides a direct approach to defining their physiological function both peripherally and in the central nervous system.     

Agonist-dependent interaction of the rat somatostatin receptor subtype 2 with cortactin-binding protein 1.

We report here an interaction between the C terminus of the rat somatostatin receptor subtype 2 (SSTR2) and a protein that has recently been identified as cortactin-binding protein 1 (CortBP1). Interaction is mediated by the PDZ (PSD-95/discs large/ZO-1) domain of CortBP1. As shown by in situ hybridization, SSTR2 and cortactin-binding protein are coexpressed in the rat brain. The association between SSTR2 and the PDZ-domain of CortBP1 was verified by overlay assays and by coprecipitation after transfection in human embryonic kidney (HEK) cells. Analysis by confocal microscopy indicates that CortBP1 is distributed diffusely throughout the cytosol in transfected cells and that it becomes concentrated at the plasma membrane when SSTR2 is present. This process is largely increased when the receptor is stimulated by somatostatin; as CortBP1 interacts with the C terminus of SSTR2, our data suggest that the binding of agonist to the receptor increase the accessibility of the receptor C terminus to the PDZ domain of CortBP1. Our data for the first time establish a link between a G-protein coupled receptor and constituents of the cytoskeleton.     

Interaction of the somatostatin receptor subtype 1 with the human homolog of the Shk1 kinase-binding protein from yeast

Interaction between the C terminus of a G-protein-coupled receptor and intracellular constituents may represent a crucial step in regulating signal transduction. To identify potential interacting candidates the C terminus of the somatostatin receptor subtype 1 was used as bait in a yeast two hybrid screen of a human brain cDNA library. We identified the human Skb1 sequence (Skb1Hs) as interacting protein, which is homologous to the yeast protein known Skb1 to down-regulate mitosis in Schizosaccharomyces pombe via binding to the Shk1 protein kinase; the latter is a homolog to the mammalian p21(cdc42/Rac)-activated protein kinases. Interaction required almost the entire C terminus of the somatostatin receptor subtype 1 including the conserved NPXXY motif of transmembrane region seven; in the case of the Skb1Hs most of the N terminus and an S-adenosylmethionine binding domain were mandatory, whereas the C terminus was not essential. Interaction was verified by coexpression experiments in human embryonic kidney cells. As revealed by immunocytochemical analysis Skb1Hs expressed alone aggregates in large cytosolic clusters. When coexpressed, receptor subtype 1 and Skb1Hs were colocalized at the cell surface; these cells showed a strong increase in somatostatin binding compared with cells expressing the receptor only. This may suggest that Skb1Hs acts like a chaperone by correctly targeting the receptor to the cell surface.     

Synthesis of substituted imidazopyrazines as ligands for the human somatostatin receptor subtype 5

A new preparation of trisubstituted imidazopyrazines and dihydroimidazopyrazines via parallel synthesis using aminoacids and bromoketones resulted in the discovery of non-peptidic sst5 selective agonists.     

Identification of proteins interacting with the rat somatostatin receptor subtype 2.

Using the yeast two hybrid system we have identified a novel protein termed somatostatin receptor interacting protein (SSTRIP) from human brain which interacts with the rat somatostatin receptor subtype 2. Interaction with the receptor C-terminus is mediated by a PSD-95/discs large/ZO-1 (PDZ) domain which exhibits high similarity to the PDZ domain of cortactin binding protein 1 (CortBP1). SSTRIP and CortBP1 define a novel family of multidomain proteins containing ankyrin repeats, SH3- and SH3 binding regions and a sterile alpha motif (SAM domain) in addition to the PDZ domain. Both SSTRIP and CortBP1 can be co-immunoprecipitated with the somatostatin receptor when co-expressed in HEK cells. Interestingly, co-localization of SSTR2 and CortBP1 at the plasma membrane is increased when SSTR2 is stimulated by agonists.     

Characterization of the H1.5 gene completes the set of human H1 subtype genes

The H1 histone family in mammals contains at least seven subtypes. In the past we have isolated six of the seven genes encoding these isoforms. To complete the set of the human H1 histone genes, we have designed two PCR primers deduced from a partially published sequence of the remaining histone H1 gene [Carozzi et al. (1984)Science 224, 1115–1118] and from a consensus sequence which we have derived from the conserved region of human histone H1 genes. Using these primers we have amplified a 417-bp DNA fragment from total human DNA. This fragment was used for screening a human phage genomic library. Two overlapping clones were isolated. The region contains a set of 5 genes representing each of the five histone classes. In continuation of our numbering of human H1 genes, we have named this H1 gene H1.5. This gene encodes a protein almost identical to the previously published protein sequence designated H1a [Ohe et al. (1986)J. Biochem. 100, 359–368]; since the changes are in a region of some uncertainty of the peptide sequencing, we conclude that the newly isolated gene codes for the H1a protein. The structures of the flanking regions of the genes except the H2B gene are typical for histone genes. They include: (1) a CCAAT element in the promotor region, (2) a TATA box and (3) a palindromic termination element. The H2B sequence shows no typical regulatory elements and no complete ORF, therefore we consider it as a pseudogene. The expression of the H1.5 gene was examined in several cell lines.