Arterioscler Thromb Vasc Biol

Arterioscler Thromb Vasc Biol. that, consistent with the postulated tumor suppressor function, overexpression of full-length leads to impaired cellular proliferation, while knockdown has the opposite effect. The recombinant construct represents a valuable tool to unravel the largely unknown physiological role of LRP1B and its potential functions in cancer pathogenesis. gene with homozygous deletions of exons or abnormal transcripts missing portions of the sequence were observed. Therefore, LRP1B was postulated as a putative tumor suppressor. In subsequent studies, LRP1B was found to Phosphoramidon Disodium Salt be inactivated in multiple Phosphoramidon Disodium Salt malignancies, namely urothelial cancer, hepatobiliary tumors, esophageal carcinoma, cervix carcinoma, glioblastoma, oral squamous cell carcinoma, small B-cell lymphoma, acute lymphoblastic leukemia, gastric cancer, thyroid cancer, melanoma, ovarian cancer, renal cell cancer, and adrenocortical carcinoma [2C15]. Besides allelic loss of heterozygosity and inactive mRNA transcripts, DNA methylation of CpG islands has been described as mechanism leading to decreased expression in various tumors [4, 8C11]. Recently, LRP1B was identified as integration site for hepatitis B computer virus and human papilloma computer virus presumably with impact on LRP1B expression [16, 17]. Taken together, these observations strongly suggest a role of LRP1B in tumorigenesis and strengthen the initial hypothesis of the receptor serving as a tumor suppressor. Recently, we have characterized the expression of LRP1B in normal human tissues, which appears to be mostly restricted to brain, skeletal muscle, thyroid gland and testis. In addition, expression in smooth muscle cells of the arterial wall has been described [18]. LRP1B is one of the largest transmembrane receptors comprising 4599 amino acids encoded by an mRNA of 13800 base pairs. Similar to the homologous LRP1 receptor, LRP1B contains four ligand binding domain name regions separated by EGF precursor homology regions, a transmembrane segment and a cytoplasmic tail made up of two NPxY motifs [1]. In contrast to the homologous LRP1, LRP1B is not cleaved by furin and therefore migrates as single polypeptide chain with an apparent molecular weight of 600 kD on SDS polyacrylamide gels [19]. To gain insight into the physiological functions of LRP1B, a knockout mouse model has been generated by replacing the transmembrane domain name (exon 88) with a neomycin cassette, resulting in the absence of a membrane-inserted receptor. These mice were viable and fertile and did not show any obvious abnormalities, including no increased tumor rate [19]. However, when the gene was inactivated by more proximal deletions, no viable homozygous mutant animals were obtained, strongly suggesting a crucial role for the extracellular domain name in normal development [20]. To further characterize the physiological function of the receptor, several attempts have been made to construct a recombinant LRP1B receptor. However, due to the Phosphoramidon Disodium Salt enormous size of the polypeptide chain, only minireceptors comprising a part of the LRP1B sequence (ligand binding domain name region IV, transmembrane segment and intracellular tail) and soluble ligand binding ectodomains have been constructed [19, 21]. In the present study we used a PCR-based strategy to construct a recombinant full-length expression vector. This recombinant receptor was then introduced into human cells lacking endogenous LRP1B and cellular proliferation was analyzed. To exclude artifacts caused by overexpression, control experiments using siRNA to silence LRP1B expression were performed. RESULTS Amplification and subcloning of N-terminal, middle and C-terminal fragments Due to the enormous size of the cDNA (13.8 kb, Genebank “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_053011″,”term_id”:”153792246″,”term_text”:”NM_053011″NM_053011), the coding sequence was divided into three parts (N-terminal (3810 bp), middle (5970 bp) and C-terminal (4020 bp) fragments) and amplified separately from mouse brain cDNA using specific primers (Determine ?(Figure1).1). To ensure efficient transcription, a Kozak consensus sequence was included preceding the start codon within the N-terminal fragment. The integrity of the sequences was confirmed by restriction enzyme digestion and complete sequencing. Even with polymerases SACS made up of proof reading enzymes, single base substitutions cannot be avoided in these large amplified DNA segments. Therefore, in several instances multiple amplified clones had to be analyzed to obtain sequences without non-conservative base exchanges. We found a particular high rate of base substitutions in a 2213 bp GC-rich region within the middle fragment.