Correlation of type I insulin-like growth factor receptor (IGF-I-R) and insulin receptor-related receptor (IRR) messenger RNA levels in tumor cell lines from pediatric tumors of neuronal origin
Introduction
The receptors of the insulin receptor family, insulin receptor (IR), type-I-IGF receptor (IGF-I-R) and insulin receptor-related receptor (IRR) are heterotetrameric (αββα) glycoproteins with highly homologous amino acid sequences [1], [2], [3], [4]. A full-length human IRR cDNA was recently cloned and sequenced [5] (accession number AF064078). IGF-I, IGF-II and insulin are able to bind to the IR and to the IGF-I-R, but not to the IRR [6]. So far, no ligand has been found for the IRR (orphan receptor). In addition its cellular function has not yet been elucidated. Interestingly, insulin was capable of stimulating the tyrosine kinase activity of a chimera containing the extracellular domain of the IR and the intracellular domain of the IRR [6], [7], [8]. Since the IR, IGF-I-R and IRR have intrinsic tyrosine kinase activity of their two membrane spanning β-subunits in common [1], [2], [3], [4], their signalling pathways are expected to show similarities.
While IR and IGF-I-R are expressed in many mammalian tissues, IRR expression seems to be restricted to a few tissues. The IRR mRNA was found to be most abundant in the sensory neurons of the trigeminal and dorsal root ganglion during the ontogenesis of rodents [2], [9], [10]. In addition, IRR mRNA is expressed in the renal distal tubule cells and focally in the stomach in the enterochromaffin-like cells [9], [11]. Importantly, IRR mRNA is detectable in neuroblastomas and, so far, the naturally occurring protein, i.e. the product encoded by the human IRR gene has only been detected in neuroblastomas [7]. Since the IRR expression of neuroblastomas was found to be related to the stage of the tumors [7], the elucidation of its biological function might be important and of clinical relevance in the area of neuroblastoma research. In addition, an association between alterations of the expression and/or signal transduction of tyrosine kinase receptors, in particular IGF-I-R, and tumor development (see Ref. [12] for review) has been suggested.
We have asked whether or not mRNA expression of the two tyrosine kinase receptors, IRR and IGF-I-R was related in human tumor cells and whether or not receptor expression pattern was tumor specific. Receptor mRNA levels were measured in parallel by semi-quantitative RT-PCR in 18 cell lines from pediatric tumors: in seven neuroblastoma cell lines and in 11 soft tissue sarcoma (STS) cell lines, five of which were of neuronal origin.
Section snippets
Cell lines and cell culture
A commercially available human fetal kidney cDNA (Marathon-Ready™-cDNA, Clontech, Germany, Lot No. 7060911) designated ‘kidney’ was used as IRR mRNA-positive template. The embryonal epithelial kidney cell line 293-O (ATCC # CRL 173; Rockville, USA) was used as a negative control for IRR. The neuroblastoma cell lines MHH-NB-11 (# DSM ACC 157), Kelly (# DSM ACC 355), and SHSY-SY (# DSM ACC 209) were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).
IRR mRNA and IGF-I-R mRNA levels in neuroblastoma cell lines
Fig. 1 illustrates the optimization of the cycle numbers required for RT-PCR of IRR and IGF-I-R using cDNA of the STS cell line A673. After RT-PCR the gels were stained with ethidium bromide and the bands analyzed densitometrically. In the case of IRR, the difference was most obvious after 40 cycles; in the case of IGF-I-R, after 18 cycles. Fig. 2 shows a typical PCR control experiment to verify the specificity of the method. Lane 1 represents the marker, lane 2 the cell line 293-0 as a
Discussion
We compared the mRNA levels of the two tyrosine kinase receptors IRR and IGF-I-R, measured by RT-PCR, in seven neuroblastoma and 11 soft tissue sarcoma cell lines. The most important results were, first, that all seven neuroblastoma cell lines and two of the five STS cell lines of neuronal origin expressed remarkable amounts of IRR mRNA. Second, IRR mRNA was found only in those cell lines which also expressed IGF-I-R mRNA. Third, IRR and IGF-I-R mRNA levels were highly correlated. In the
Acknowledgements
We thank Katja Simon-Klingenstein, Olga Hospital Stuttgart for her excellent technical assistance with the soft tissue sarcoma cell lines. Supported in part by Mildred-Scheel Stiftung, Deutsche Krebshilfe, Bonn; Grant Ki 1000, Bonn, Germany by the Growth Research Center, Tübingen Germany and by grant El 167/3-1 from the Deutsche Forschungsgemeinschaft (DFG).
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2005, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Since its discovery in 1989 by Shier and Watt [10] and the publication of the full-length sequence [20], different approaches have been employed to reveal the function of the insulin receptor-related receptor, mainly impeded by the fact that the IRR still has to be regarded as an orphan receptor with unknown natural ligand. Although triple-gene knockout experiments in mice suggest that IRR might be involved in testis determination [14], this finding does not explain its prevalent expression in neuronal, pancreatic or kidney cells [9,15,29–33]. Because of the great extent of sequence homology of the IRR to the two other members of the insulin receptor family, the insulin receptor (InsR) and the IGF-I receptor (IGF-IR), especially within the cytoplasmic portions of the receptors, we employed a yeast two-hybrid approach to investigate the signaling capabilities of the IRR.
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