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Identification and investigations of leucine-rich repeats and immunoglobulin-like domains protein 2 (LRIG2)
Umeå University, Faculty of Medicine, Department of Radiation Sciences.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Umeå: University , 2010. , 52 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1346
Keyword [en]
LRIG, PDGF, oligodendroglioma
National Category
Medical and Health Sciences
Research subject
Oncology
Identifiers
URN: urn:nbn:se:umu:diva-33784ISBN: 978-91-7459-011-1 (print)OAI: oai:DiVA.org:umu-33784DiVA: diva2:318137
Public defence
2010-05-28, Sal 244 Lionssalen, By 7, Norrlands universitetssjukhus, Umeå, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2010-05-10 Created: 2010-05-06 Last updated: 2010-05-10Bibliographically approved
List of papers
1. Characterization and tissue-specific expression of human LRIG2
Open this publication in new window or tab >>Characterization and tissue-specific expression of human LRIG2
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2004 (English)In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 332, 35-43 p.Article in journal (Refereed) 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.

Keyword
Leucine-rich repeats and immunoglobulin-like domains, LRIG1, Lig-1
National Category
Medical and Health Sciences
Research subject
Oncology
Identifiers
urn:nbn:se:umu:diva-15347 (URN)10.1016/j.gene.2004.02.002 (DOI)15145052 (PubMedID)
Available from: 2007-09-13 Created: 2007-09-13 Last updated: 2010-05-10Bibliographically approved
2. Lrig2 knock-out mice have increased mortality, impaired fertility and transiently reduced body weight
Open this publication in new window or tab >>Lrig2 knock-out mice have increased mortality, impaired fertility and transiently reduced body weight
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(English)Manuscript (preprint) (Other academic)
National Category
Cancer and Oncology
Research subject
Oncology
Identifiers
urn:nbn:se:umu:diva-32613 (URN)
Available from: 2010-03-18 Created: 2010-03-18 Last updated: 2010-05-10Bibliographically approved
3. Cytoplasmic LRIG2 expression is associated with poor oligodendroglioma patient survival.
Open this publication in new window or tab >>Cytoplasmic LRIG2 expression is associated with poor oligodendroglioma patient survival.
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2009 (English)In: Neuropathology (Kyoto. 1993), ISSN 0919-6544, E-ISSN 1440-1789, Vol. 29, no 3, 242-247 p.Article in journal (Refereed) Published
Abstract [en]

The three leucine-rich repeats and immunoglobulin-like domains (LRIG) genes encode integral membrane proteins. Of these, LRIG1 negatively regulates growth factor signaling and is implicated as a tumor suppressor in certain malignancies. In astrocytic tumors, the subcellular distribution of LRIG proteins is associated with specific clinicopathological features and patient survival. The role of LRIG proteins in oligodendroglioma has not previously been studied. Here we used immunohistochemistry to analyze the expression of the LRIG proteins in 63 oligodendroglial tumors, and evaluated possible associations between LRIG protein expression and clinicopathological parameters. Notably, cytoplasmic LRIG2 expression was found to be an independent prognostic factor associated with poor oligodendroglioma patient survival. This is the first report of an LRIG protein showing a negative effect on survival, suggesting that LRIG2 might have a function different from that of LRIG1, and possibly contributing to the etiology of oligodendroglioma.

Keyword
immunohistochemistry, LRIG proteins, LRIG2, oligodendroglioma, patient survival
National Category
Cancer and Oncology
Research subject
Oncology
Identifiers
urn:nbn:se:umu:diva-11524 (URN)10.1111/j.1440-1789.2008.00970.x (DOI)18992012 (PubMedID)
Available from: 2009-01-13 Created: 2009-01-13 Last updated: 2011-09-02Bibliographically approved
4. LRIG2 promotes PDGF induced experimental glioma
Open this publication in new window or tab >>LRIG2 promotes PDGF induced experimental glioma
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(English)Manuscript (preprint) (Other academic)
National Category
Cancer and Oncology
Research subject
Oncology
Identifiers
urn:nbn:se:umu:diva-32614 (URN)
Available from: 2010-03-18 Created: 2010-03-18 Last updated: 2010-05-10Bibliographically approved

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Holmlund, Camilla

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