Arrows point to the two major 60 and 75 kD bands after glycosidase treatment; and to a putative SEA cleavage product (in dotted box)

Arrows point to the two major 60 and 75 kD bands after glycosidase treatment; and to a putative SEA cleavage product (in dotted box). Because GPR110 contains 19 predicted N-linked glycosylation sites, the bands running at 100 and 80 kD fall short of the expected size. mass of about 100 kD. Isoforms 2 and 3 are truncated products of isoform 1, and are 25 and 23 kD, respectively. These truncated isoforms lack the seven-span transmembrane domain name characteristic of GPR proteins and thus are not likely to be membrane anchored; indeed, isoform 2 can be secreted. Compared with the median gene expression of ~200 selected genes, GPR110 expression was low in most tissues. However, it experienced higher than average gene expression in normal kidney tissue and in prostate tissues originating from older donors. Although identified as an oncogene in murine T lymphomas, GPR110 is usually greatly overexpressed in CF53 human lung and prostate cancers. As detected by immunohistochemistry, GPR110 was overexpressed in 20 of 27 (74%) lung adenocarcinoma tissue cores and in 17 of 29 (59%) prostate adenocarcinoma tissue cores. Additionally, staining with a GPR110 antibody enabled us to differentiate between benign prostate hyperplasia and potential incipient malignancy. Conclusion Our work suggests a role for GPR110 in tumor physiology and supports it as a potential therapeutic candidate and disease marker for both lung and prostate malignancy. Background GPCRs are seven transmembrane receptors that vary extensively in their biological functions. Upon ligand binding, these receptors transduce a signal via a G protein. This fact has been used extensively in pharmacology to select inhibitors of biological pathways. A large portion of all drugs currently on the market target GPCRs. Drugs targeting users of this integral membrane protein superfamily represent the core of modern medicine [1]. There are numerous so-called orphan receptors–receptors without a CF53 known ligand, a known signaling pathway, or a known function. Despite the lack of information, one can presume that orphan receptors have important biological roles. One of these orphan receptors is usually GPR110, about which little is known other than its gene structure and potential isoforms that can be inferred from published transcript data. In a large murine retroviral mutagenesis screen, we recognized GPR110 as an oncogene. The GPR110 protein contains two protein domains where cleavage can potentially occur: the SEA domain and the GPS domain. Self-cleavage has been reported for the SEA domain in human MUC1 [2] and in rat Muc3 [3]. According to these reports, the cleaved SEA product reassociates with the membrane-bound CF53 protein by noncovalent interactions. Cleavage at the GPS domain was first exhibited in the GPCR latrophilin [4]. Cleaved products of an overexpressed GPCR might be found in the blood, which could serve as an easily accessible clinical marker. Furthermore, alternatively spliced isoforms that are not membrane anchored may instead be potentially secreted and also be found in the blood. The rich possibility of GPR110 as a therapeutic candidate and diagnostic marker led us to study the synthesis of its numerous isoforms and to survey human cancers for its overexpression. Methods Cloning and tagging of GPR110 isoforms GPR110 isoforms 1 and 2 were amplified from PC-3 cDNA using a set of primers designed to their common 5′ UTR and their respective 3′ UTR MADH9 regions. Forward primer 5′-CACCAGTCACAGACTATGC-3′ and reverse primer 5′-ACCCGATCGAATACTGAGC-3′ (isoform 1, 3′ UTR) and reverse primer 5′-CAGGGGAATCTCTTGAACCCG-3′ (isoform 2, 3′ UTR). Products from the first PCR reactions were used as themes in a nested PCR with the following primers: forward primer 5′-TTCGGTACCACCATGAAAGTTGGAGTGC-3′ (110_F_Kpn), reverse primer 5′-CCCTCTAGATTATTCATTTGAGACAAACTG-3′ (isoform 1, with quit codon) and reverse primer 5′-CCTTCTAGAGATTGTGCCATTGCACTC-3′ (isoform 2, no quit codon). The PCR products were then cloned into pcDNA3.1(+) (Invitrogen) using em Kpn /em I and em Xba /em I restriction sites to make constructs pcDNA/Iso1 and pcDNA/Iso2. Sequences of these clones matched published RefSeq sequences on NCBI. GPR110 isoform 3 with no quit codon was amplified from pcDNA/Iso1 using the primers 110_F_Kpn and reverse primer 5′-CCCTCTAGACCGAAATTGGGTGACC-3′. A version of isoform 1 with no quit codon was amplified from your pcDNA/Iso1 construct using primer 110_F_Kpn and reverse primer 5′-CCCTCTAGATTCATTTGAGACAAACTGAG-3′. Isoforms 1-3 made up of no stop codons were then cloned into a version of pcDNA3.1(+) containing the HA epitope between restriction sites em Xba /em I and em Apa /em Ion the pcDNA3.1(+) vector creating constructs Iso1-HA, Iso2-HA, and Iso3-HA. Three additional HA-tagged versions of isoform 1 were made using pcDNA/Iso1 as CF53 a template with the QuikChangeII Site-Directed Mutagenesis Kit (Stratagene). The following primers were utilized for the three constructs: HA466: 5′-TTAGAATTATCAGAGCAAAGTACCCATACGATGTTCCAGATTACGCTACCACAGACTGCAACAG-3′ and 5′-CTGTTGCAGTCTGTGGTAGCGTAATCTGGAACATCGTATGGGTACTTTGCTCTGATAATTCTAA-3′; HA1036: 5′-CCTGCAGCAGTGGCTACCCATACGATGTTCCAGATTACGCTAGGGGAAACATCACAGC-3′ and 5′-GCTGTGATGTTTCCCCTAGCGTAATCTGGAACATCGTATGGGTAGCCACTGCTGCAGG-3′; and HA1393: 5′-GTCTTACTGCGGGAAGAAAAGTACCCATACGATGTTCCAGATTATGCCAGCTCACG-3′ and 5′-CGTGAGCTGGCATAATCTGGAACATCGTATGGGTACTTTTCTTCCCGCAGTAAGAC-3′. Construct HA466 has a HA tag located N-terminal to the SEA domain name whereas HA1036 and.

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