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Characterization and tissue-specific expression of human LRIG2
Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper, Onkologi.
Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper, Onkologi.
Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM). Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper, Onkologi.
Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper, Onkologi.
Visa övriga samt affilieringar
2004 (Engelska)Ingår i: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 332, s. 35-43Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

We have recently identified and cloned the human LRIG1 gene (formerly LIG1). LRIG1 is a predicted integral membrane protein with a domain organization reminiscent of the Drosophila epidermal growth factor (EGF)-receptor antagonist Kekkon-1. We have searched for additional members of the human LRIG family and identified LRIG2 (KIAA0806). The LRIG2 gene was localized to chromosome 1p13 and had an open reading frame of 1065 amino acids. The LRIG2 protein was predicted to have the same domain organization as LRIG1 with a signal peptide, an extracellular part containing15 leucine-rich repeats and three immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tail. The LRIG2 amino acid sequence was 47% identical to human LRIG1 and mouse Lrig1 (also known as Lig-1). Northern blotting and RT-PCR revealed LRIG2 transcripts in all tissues analyzed. Quantitative real-time RT-PCR showed the most prominent RNA expression in skin, uterus, ovary, kidney, brain, small intestine, adrenal gland, and stomach. Immunoblotting of COS-7 cell lysates demonstrated that heterologously expressed human LRIG2 had an apparent molecular weight of 132 kDa under reducing gel-running conditions. N-glycosidase F treatment resulted in a reduction of the apparent molecular weight to 107 kDa, showing that LRIG2 was a glycoprotein carrying N-linked oligosaccharides. Cell surface biotinylation experiments and confocal fluorescence laser microscopy demonstrated expression of LRIG2 both at the cell surface and in the cytoplasm. LRIG2 was detected in tissue lysates from stomach, prostate, lung, and fetal brain by immunoblotting. In conclusion, LRIG2 was found to be a glycoprotein which was encoded by a gene on human chromosome 1p13 and its mRNA was present in all tissues analyzed.

Ort, förlag, år, upplaga, sidor
2004. Vol. 332, s. 35-43
Nyckelord [en]
Leucine-rich repeats and immunoglobulin-like domains, LRIG1, Lig-1
Nationell ämneskategori
Medicin och hälsovetenskap
Forskningsämne
onkologi
Identifikatorer
URN: urn:nbn:se:umu:diva-15347DOI: 10.1016/j.gene.2004.02.002PubMedID: 15145052OAI: oai:DiVA.org:umu-15347DiVA, id: diva2:155019
Tillgänglig från: 2007-09-13 Skapad: 2007-09-13 Senast uppdaterad: 2018-06-09Bibliografiskt granskad
Ingår i avhandling
1. The LRIG-family: identification of novel regulators of ErbB signaling with clinical implications in astrocytoma
Öppna denna publikation i ny flik eller fönster >>The LRIG-family: identification of novel regulators of ErbB signaling with clinical implications in astrocytoma
2006 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Astrocytic tumors are the most common malignancies of the central nervous system, accounting for more than 60% of all primary brain tumors. The prognosis for high grade astrocytoma patients is dismal and there is no curative treatment, today. A molecular hallmark of astrocytic tumors is dysregulated receptor tyrosine kinase signaling, especially of the epidermal growth factor receptor (EGFR, ErbB1). The aim of the present thesis was to identify endogenous human proteins that downregulate the function of the ErbB1 receptor. We identified a human gene family, the leucine-rich repeats and immunoglobulin-like domains family (LRIG), consisting of LRIG1, LRIG2 and LRIG3, which might fulfill this criterion.

Two candidates were identified, LRIG1 and LRIG2, which genes were localized to regions frequently deleted in human cancers, chromosome bands 3p14 and 1p13, respectively. LRIG1 and LRIG2 mRNA were expressed in all tissues analyzed, with high expression in brain and other organs. The LRIG mRNA were predicted to encode integral membrane proteins. Antibodies against LRIG1 and LRIG2 were developed and the protein expression was analyzed. LRIG1 was found to have an apparent molecular weight of 143 kDa and was expressed in most tissues with high expression in glandular tissues of the breast and prostate, and the peptic cells of the stomach. LRIG2 was slightly smaller and had an apparent molecular weight of 132 kDa. The LRIG proteins were localized to the cell surface and to perinuclear structures, where LRIG1 co-localized with the trans-Golgi network and early endosomes.

LRIG1 was found to restrict growth factor signaling by enhancing receptor ubiquitylation and degradation. We showed that LRIG1 interacted with all four members of the ErbB family and induced their downregulation. The interaction with ErbB1 involved both the LRR-domains and the Ig-like domains of LRIG1. LRIG1 enhanced receptor degradation by recruiting the E3 ubiquitin ligase c-Cbl to the LRIG1-ErbB1 complex.

LRIG1, LRIG2, and LRIG3 were expressed in glioma cell lines and tumor tissues. The LRIG expression was analyzed in 404 astrocytic tumor samples. We found that perinuclear LRIG protein expression correlated with increased survival of patients with astrocytic tumors. Especially perinuclear LRIG3 showed strong correlations with patient survival and tumor cell proliferation index.

In summary, this thesis contains the discovery and characterization of human LRIG1 and LRIG2. LRIG1 was found to interact with ErbB receptors and downregulate their function. In a clinical material, expression of LRIG proteins correlated with survival in patients with astrocytic tumors.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå universitet, 2006. s. 41
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1027
Nyckelord
astrocytic tumors, EGFR, ErbB, glioblastoma multiforme, LRIG, negative regulation.
Nationell ämneskategori
Cancer och onkologi
Identifikatorer
urn:nbn:se:umu:diva-783 (URN)91-7264-074-X (ISBN)
Disputation
2006-05-24, 244 Lionssalen, By 7, Norrlands universitetssjukhus, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2006-05-03 Skapad: 2006-05-03 Senast uppdaterad: 2018-06-09Bibliografiskt granskad
2. The Identification and Characterisation of LRIG Gene Family and Its Expression in Astrocytic Tumours
Öppna denna publikation i ny flik eller fönster >>The Identification and Characterisation of LRIG Gene Family and Its Expression in Astrocytic Tumours
2004 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Gliomas are the most common primary brain tumours, and their capacity to invade surrounding normal brain prevents complete removal of the tumour. Malignant glioma has still a poor prognosis. However, with the rapid development of molecular biology our understanding about glioma has increased dramatically. Among known growth factors, EGF and its receptor are frequently amplified and over expressed in malignant glioma. Therefore, it is of interest to find approaches to hamper the activity of EGF/EGFR. The aim of this thesis was to identify and characterize human analogues to a recently identified gene in Drosophilia, kekkon-1, which negatively regulates the activity of Drosophilia EGF receptor.

In the first part, we set up a quantitative real-time RT-PCR assay, which showed good linearity, reproducibility and uniformity. We analyzed the expression of the most commonly used reference genes, and showed that 18S was the most reliable endogenous reference gene in this study.

In the second part, we cloned, identified, and sequenced a gene family, which we named leucine-rich repeats and immunoglobulin–like domains family (LRIG). The LRIG gene family had three vertebrate paralogs and one homolog in ascidiacea. The proteins encoded by human LRIG genes shared an overall structure with a signal peptide, 15 tandems leucine-rich repeats with N- and C- terminal flanking regions followed by 3 immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tail. Northern blot showed the mRNA sizes to be 5.5 kb for LRIG1, 4.8 kb for LRIG2, and 5.1 kb for LRIG3. LRIG1-3 mRNAs were detected in all human and mouse tissues analyzed, however, at various levels. FISH and BLAST analysis showed that LRIG1 was located at 3p14, LRIG2 at 1q13, and LRIG3 at 12q13. LRIG1 was shown to be down-regulated in several cancer cell lines and proposed to be a tumour suppressor gene.

In the third part, we analysed the expression of LRIG gene family in human astrocytic tumours. LRIG1-3 mRNAs were detected in all human glioma cell lines, in primary tumour tissues and control-matched normal brain tissues, at various levels. Subcellular localizations of LRIG1-GFP fusion proteins were visualized in nuclear, perinuclear, and cytoplasmic compartment. According to the predicted protein sequences, short peptides were synthesized and used to raise antibodies in rabbits. The antibodies were used for immunohistochemical analysis of LRIG1-3 in 404 human astrocytic tumours in a tissue micro array. The pattern of immunoreactivity of LRIG1-3 was heterogeneous with staining in nuclear, perinuclear and cytoplasmic compartment of positive tumour cells. Perinuclear staining of LRIG1-3 displayed a significant inverse correlation with WHO grade and especially positive LRIG3 perinuclear and cytoplasmic staining correlated with a low proliferation index. The LRIGs correlated with survival, and LRIG3 perinuclear staining was in addition to tumour grade an independent prognostic factor. The results suggest that LRIGs may play a role in normal tissue, and may be of importance in the pathogenesis and prognosis of tumours. The exact function of LRIG1-3 remains to be established.

Förlag
s. 61
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 932
Nyckelord
astrocytoma, brain, EGFR, LRIG, leucine-rich repeat, real-time RT-PCR
Forskningsämne
onkologi
Identifikatorer
urn:nbn:se:umu:diva-370 (URN)91-7305-771-1 (ISBN)
Disputation
2004-12-11, Lionsalen, Onkologi, Norrlands universitetssjukhus, Umeå, 11:00 (Engelska)
Opponent
Tillgänglig från: 2004-11-18 Skapad: 2004-11-18 Senast uppdaterad: 2018-03-15Bibliografiskt granskad
3. Identification and investigations of leucine-rich repeats and immunoglobulin-like domains protein 2 (LRIG2)
Öppna denna publikation i ny flik eller fönster >>Identification and investigations of leucine-rich repeats and immunoglobulin-like domains protein 2 (LRIG2)
2010 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Receptor tyrosine kinases (RTKs) constitute a family of proteins controlling cell growth and proliferation and whose activities are tightly controlled in normal cells. LRIG1 is a negative regulator of RTK signaling and is a proposed tumor suppressor. The aim of this thesis was to identify and study possible paralogs of LRIG1. By using the basic local alignment search tool and cDNA cloning, a human mRNA sequence with similarity to LRIG1 was identified and named LRIG2. By fluorescence in situ hybridization analysis, LRIG2 was found to reside on chromosome 1p13. The LRIG2 amino acid sequence was 47% identical to LRIG1, and the predicted protein domain organization was the same as that of LRIG1. Antibodies against LRIG2 were developed and the apparent molecular weight of the protein was determined to be 132 kDa by SDS-polyacrylamide gel electrophoresis and Western blot analysis. The sub-cellular localization was studied by cell surface biotinylation experiments and confocal fluorescence laser microscopy, which revealed that LRIG2 resided at the cell surface and in the cytoplasm.

The expression patterns of LRIG2 mRNA, during development and in adult tissues, were evaluated using whole-mount in situ hybridization and quantitative real-time RT-PCR, respectively. In E10.5, E11.5 and E12.5 mouse embryos, the Lrig2 expression domains were both overlapping and unique as compared to the expression domains of Lrig1 and the third family member, Lrig3. In adult human tissues, the most prominent LRIG2 mRNA expression was found in skin, uterus and ovary. To study the developmental and physiological role of LRIG2, Lrig2 knock-out mice were generated. The knock-out mice were born at Mendelian frequencies without any apparent morphological abnormalities. However, Lrig2 knock-out mice showed reduced body weight between 5 days and 12-15 weeks of age, increased mortality, and impaired reproductive capacity.

To study the role of LRIG2 as a prognostic factor in oligodendroglioma, LRIG2 expression was analyzed in 65 human oligodendrogliomas by immunohistochemistry. Cytoplasmic LRIG2 expression was an independent prognostic factor associated with poor oligodendroglioma patient survival. The possible functional role of LRIG2 in oligodendroglioma biology was further investigated using the RCAS/tv-a mouse model. Tumors resembling human oligodendroglioma were induced by intracranial injection of PDGFB carrying RCAS retroviruses into newborn Ntv-a mice. Lrig2 wild-type animals developed tumors at a higher frequency and of higher malignancy than the Lrig2 knock-out mice. This result supports the notion that LRIG2 promotes PDGF-induced oligodendroglioma genesis. A possible molecular mechanism was revealed as LRIG2 overexpression increased PDGFRa levels in transfected cells. In summary, we identified a new gene named LRIG2, showed that it is expressed in a variety of tissues during development and in adulthood, knocked it out and found that it was required for proper animal growth, health, and reproduction. We also found that Lrig2 expression promoted PDGF-induced oligodendroglioma genesis and was associated with poor oligodendroglioma patient survival, possibly via a PDGFRa stabilizing function.

Ort, förlag, år, upplaga, sidor
Umeå: University, 2010. s. 52
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1346
Nyckelord
LRIG, PDGF, oligodendroglioma
Nationell ämneskategori
Medicin och hälsovetenskap
Forskningsämne
onkologi
Identifikatorer
urn:nbn:se:umu:diva-33784 (URN)978-91-7459-011-1 (ISBN)
Disputation
2010-05-28, Sal 244 Lionssalen, By 7, Norrlands universitetssjukhus, Umeå, 09:00 (Svenska)
Opponent
Handledare
Tillgänglig från: 2010-05-10 Skapad: 2010-05-06 Senast uppdaterad: 2018-06-08Bibliografiskt granskad

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Holmlund, CamillaNilsson, JonasGolovleva, IrinaHenriksson, RogerHedman, Håkan

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