ILZRp, IL3R, IL4R, ILGR, IL7R, granulocyte-macrophagc colony stimulating factor (GMCSFR), granulocyte colony stimulating factor (GCSFR), prolactin (PRLR), growth hormone (GHR), and crythropoictin (EPOR) [l-3], These receptors possess a single hydrophobic transmembrane area, a highly variable cytoplasmic domain -both in length and sequence -and an cxtracellular domain with a stretch of about 200 amino acids, here named D200, which is significantly conserved, although at a relatively low level (1%3SVo) [I]. IL3R has a tandem of two such domains. For GCSFR, three fibronectin type III domains have been recognized hctween the D200 domain and the transmembranc segment. One additive segment occurs at the Nterminal part of ILBR and of GCSFR and has been recognized to be immunoglobulin like [2,4] On the other hand, Interferon Receptors (INFRs) show similar organization, either with a unique D200-like domain for they (INFgR) The recognition of conserved features for these biologically related proteins could help the understanding of their mechanisms of action and might open perspectives for therapeutic applications.In this paper we describe a two-dimensional (2D) sequence analysis of the extraceliular hormone binding domain of these receptors. We show that the D200 amino acid conserved domains possess internal symmetry and so probably result from the duplication of a subdomain = 100 amino acids long, here named SDlOO,4 and SDlOOB. Further, we show that GMCSFR comprises an additional SD100 subdomain at its Nterminal part, These subdomains appear similar to and have the same size as the highly repeated type III domains of fibronectin, which has already been shown to be sequence related to the here-named SDIOOB [7]. We also confirm and expand the homology with repeated domains of contactin [8] RESULTSVisual inspection of WCA plots of the D200 conserved extracellular domains of CRs and INFRs families led to the frequent observation of clear cut loop (or hinge) regions in their middle (EPDPP for rbPRLR, QPDPP for huGHR, QPPPPKD for muIL3R first D200 domain, KPLAPD for muIL4R, QPDPPAN for huILBR, GPPE for huINFaR first D200 domain) (see the linear alignment reported in Fig. 3A) similar to that noted between D200 domains, e.g. PPPEN for huINFaR [5]. So a major interrupter of folding could be suspected to be present. Further, HCA plots of many receptors suggest (qualitatively and quantitatively) a structural homology between these two equivalent sized halfr; of the D200 domains. Fig. 1 exnmplifics this with the muEPOR where the middle breaker is DAPAG around position 120. For the two halves of the DZOO domain (SDlOOA and SDlQOB) OF that. receptor, HCA-score and the rcsulranc alignment sequence identity arc 72% and 13%, respectively. These values arc within the range of significant homology otherwise observed for . A test set of all SD100 co&n-parisons (12 SDlOO, 66 pairs in all) for muEPOR, muIL3R, huIL7R, huINFaR shows that among previously known corresponding sequences (SDlOOA/SDlOOA and SDlOOB/SDlOOB) 16 pairs have s...

Background: Suppressor of cytokine signaling (SOCS) proteins comprise a protein family, which has initially been described as STAT induced inhibitors of the Jak/Stat pathway. Recent in vivo and in vitro studies suggest that SOCS proteins are also implicated in cancer. The STAT5 induced IGF-I acts as an endocrine and para/autocrine growth and differentiation factor in mammary gland development. Whereas high levels of circulating IGF-I have been associated with increased cancer risk, the role of autocrine acting IGF-I is less clear. The present study is aimed to elucidate the clinicopathological features associated with SOCS1, SOCS2, SOCS3, CIS and IGF-I expression in breast cancer.

Two Agtll clones containing fragments of cDNA encoding the prolactin receptor from rabbit mammary gland were isolated using a rat liver prolactin receptor cDNA probe. An 1848-base-pair open reading frame encodes a mature prolactin-binding protein of 592 amino acids that contains three domains: (i) the extracellular, amino-terminal, prolactin-binding region of 210 residues; (ii) the transmembrane region of 24 residues; and (iii) the intracellular, carboxyl-terminal domain of 358 residues. This latter domain is much longer than the cytoplasmic domain (57 residues) previously described for the rat liver prolactin receptor. In addition, the sequence identity of this form of prolactin receptor with the growth hormone receptor is extended in the cytoplasmic domain.The anterior pituitary hormone prolactin (PRL) is involved in an impressive list of biological actions in all vertebrates (1) and specific receptors for this hormone have been identified in numerous organs (2). PRL is structurally related to growth hormone (GH) and chorionic somatomammotropic or placental lactogen, which are encoded by the same gene family (3). In mammals, PRL is primarily responsible for the development of the mammary gland and lactation. It stimulates the expression of milk protein genes by increasing both gene transcription and mRNA half-life (4). These actions are initiated by an interaction of PRL with specific, high-affinity receptors located in cell membranes. This receptor has been partially purified from rabbit mammary gland (5) and monoclonal antibodies against it have been developed (6). After binding of PRL to its receptors, the mechanism(s) by which the hormonal signal is transferred inside the cell remains unknown. We have shown (7) that one monoclonal antibody against the PRL receptor is a potent agonist of PRL actions in mammary cells, suggesting that PRL itself is probably not necessary beyond its binding and that the receptor is able, under certain experimental conditions, to operate alone probably by coupling to a second messenger system. However, as is true for GH, none of the classical second messengers (cAMP, cGMP, inositol phospholipids, or calcium ions) appears to be involved in PRL signal transduction (8). The knowledge of the primary structure of the receptor and the identification of functional domains may facilitate a better understanding of these phenomena. To that end, we have previously cloned cDNAs of PRL receptor from rat liver cDNA libraries (9), deduced primary structure of the protein, and established that this receptor has regions of strong sequence homology with the GH receptor, also cloned (10), but contains a much shorter cytoplasmic domain. The mammary gland is a major target organ for PRL, where in contrast to the liver, biological effects are well established (4). We have reported (9) that the major mRNA species for the PRL receptor is much larger in the mammary gland [4 kilobases (kb)] than in the liver (2.2 kb), suggesting heterogeneity in the coding sequence or in the untranslated region ...

Proteolytic degradation of ribosomal proteins occurs during the preparation of subunits of the cytoplasmic ribosomes of the protozoa Tetrahymenu thernioplrila and the isolated subunits are inactive. Addition of 5 mM iodoacetamide to cell suspensions before extraction inhibits proteolytic activity and permits isolation of active subunits. The protein complements of these subunits have been characterized in two different two-dimensional elcctrophoretic systems, and their molecular weights have been determined.Limited degradation of ribosomal proteins has been observed during isolation of ribosomes and ribosomal subunits from a variety of eukaryotic cells [l -61. This phenomenon was not observed in our earlier studies [7] with ribosomes of an amicronucleate Tetraliymeiza strain of unknown phenoset (Tetrahyniena 'pyr{forn~is' CGL) but has subscquently been shown to occur during preparation of ribosomes from several other amicronucleate strains [XI (and B. Petridou and F. Hayes, unpublished results). It also occurs, as shown here, during preparation of ribosomes from the micronucleate strain Tetruhymenu thermophila and leads to isolation of inactive ribosomal subunits unless protease inhibitors are present throughout the preparation process. Of a number of inhibitors tested, iodoacetamide was found to be the most effective. 40s and 60s ribosomal subunits prepared from T . thermophila in its presence are active in vitro and retain their activity during storage at low tcmperature. Alkylation of a small number of proteins in these subunits revealed by experiments with radioactive iodoacetamide appears not to impair their capacity to reassociate or to catalyse polyphenylalanine synthesis in vitro. The protein complements of active and inactive 40s and 60s ribosomal subunits of T. thernmphilu prepared in the presence and absence respectively of iodoacetamide have been characterized by polyacrylamide gcl electrophoresis in two different systems. The molecular weights of the proteins of active subunits have been estimated by SDS gcl electrophoresis.

Once daily milking (ODM) induces a reduction in milk production when compared to twice daily milking (TDM). Unilateral ODM of one udder half and TDM of the other half, enables the study of underlying mechanisms independently of inter-individual variability (same genetic background) and of environmental factors. Our results show that in first-calf heifers three CpG, located 10 kb upstream from the CSN1S1 gene were methylated to 33, 34 and 28%, respectively, after TDM but these levels were higher after ODM, 38, 38 and 33%, respectively. These methylation levels were much lower than those observed in the mammary gland during pregnancy (57, 59 and 50%, respectively) or in the liver (74, 78 and 61%, respectively). The methylation level of a fourth CpG (CpG4), located close by (29% during TDM) was not altered after ODM. CpG4 methylation reached 39.7% and 59.5%, during pregnancy or in the liver, respectively. CpG4 is located within a weak STAT5 binding element, arranged in tandem with a second high affinity STAT5 element. STAT5 binding is only marginally modulated by CpG4 methylation, but it may be altered by the methylation levels of the three other CpG nearby. Our results therefore shed light on mechanisms that help to explain how milk production is almost, but not fully, restored when TDM is resumed (15.1±0.2 kg/day instead of 16.2±0.2 kg/day, p<0.01). The STAT5 elements are 100 bp away from a region transcribed in the antisense orientation, in the mammary gland during lactation, but not during pregnancy or in other reproductive organs (ovary or testes). We now need to clarify whether the transcription of this novel RNA is a consequence of STAT5 interacting with the CSN1S1 distal region, or whether it plays a role in the chromatin structure of this region.