Selective Amplification And Cloning Of Four New Members Of The G Protein-coupled Receptor Family

Abstract

An approach based on the polymerase chain reaction has been devised to clone new members of the family of genes encoding guanosine triphosphate-binding protein (G protein)-coupled receptors. Degenerate primers corresponding to consensus sequences of the third and sixth transmembrane segments of available receptors were used to selectively amplify and clone members of this gene family from thyroid complementary DNA. Clones encoding three known receptors and four new putative receptors were obtained. Sequence comparisons established that the new genes belong to the G protein-coupled receptor family. Close structural similarity was observed between one of the putative receptors and the 5HT1a receptor. Two other molecules displayed common sequence characteristics, suggesting that they are members of a new subfamily of receptors with a very short nonglycosylated (extracellular) amino-terminal extension.

Identification of G protein‐coupled receptors by RNase H‐mediated hybrid depletion using Xenopus laevis oocytes as expression system

“…These include functional expression of cloned receptor cDNA in frog oocytes, hybrid selection of receptor-encoding mRNAs with cloned cDNA and expression of transfected receptor genes in cell culture followed by identification using a cell sorter (reviewed in [2]). Especially powerful has been the exploitation of known similarities between the family of G protein-coupled-receptors to construct degenerate oligonucleotide primers for the PCRmediated amplification of putative new family members [3]. A problem of this approach is that the amplified receptor fragments do not possess all the information required to encode a functional receptor and must thus be used to isolate a full-size clone from a suitable cDNA library.…”

“…In addition to their structural similarity, there is a notable degree of amino acid sequence conservation, especially within the putative transmembrane domains. Based upon this observation, degenerate oligonucleotide pools deduced from the third and sixth transmembrane domains have been used as primers in the polymerase chain reaction [3]. Using this approach, several putative members of this receptor superfamily have been successfully amplified from either genomic DNA or cDNA [3] (and unpublished data).…”

Identification of mRNAs differentially-expressed between benign and malignant breast tumour cells

“…For example, overexpression of Cyclin D1 has been shown to distinguish breast carcinomas and in situ breast lesions from benign lesions (WeinstatSaslow et al, 1995). Amongst other cDNAs up-regulated in the malignant cell library, were those corresponding to mRNAs for a putative G-protein-coupled receptor (Libert et al, 1989), for proteins associated with the mitotic spindle checkpoint, BUB3 (Brady and Hardwick, 2000) and with chromatin remodelling (Wang et al, 1996;Zhang et al, 1998;Vignali et al, 2000). Two cDNAs (LIM kinase and BUB3), which are differentially-expressed in the present malignant library, have also been found to be differentially expressed in a subtracted library representing cDNAs expressed in a pathologically homogeneous breast carcinoma specimen subtracted with cDNA from 50 000 ductal-carcinoma-in-situ cells microdissected from the surrounding normal tissue (Liu, 2001).…”

Fatty Acid Binding Receptors and Their Physiological Role in Type 2 Diabetes

“…Historically, new GPCRs were identified using homologyscreening approaches, such as low-stringency hybridization [27 -28], degenerate polymerase chain reaction (PCR) [28], or bioinformatic analyses of the genomes. The orphan GPCRs that were identified based on their homology screening, lack pharmacological identities and natural ligands.…”