The novel G-protein coupled receptor SALPR shares sequence similarity with somatostatin and angiotensin receptors

doi:10.1016/S0378-1119(00)00123-2

Gene

Volume 248, Issues 1–2, 2 May 2000, Pages 183-189

https://doi.org/10.1016/S0378-1119(00)00123-2 Get rights and content

Abstract

A cDNA encoding a novel G-protein coupled receptor (GPCR) was isolated from a human cerebral cortex cDNA library by low stringency hybridization screening. This putative seven-transmembrane domain receptor of 469 amino acids was designated SALPR ( $S$ omatostatin- and $A$ ngiotensin- $L$ ike $P$ eptide $R$ eceptor). SALPR shares the highest amount of amino acid similarity with the somatostatin (35% with SSTR5) and angiotensin receptors (31% with AT1). Reverse transcription–polymerase chain reaction (RT–PCR) analysis revealed that the SALPR mRNA is predominantly expressed in human brain regions, particularly the substantia nigra and pituitary, although the mRNA can also be detected in the peripheral tissues, albeit at low levels. Chromosomal mapping by radiation hybrid analysis localized the human SALPR gene to the chromosome 5p15.1–5p14. Transient expression of SALPR in COS-1 cells did not produce any binding sites for somatostatin or angiotensin II, indicating the necessity for further study to discover its ligand and physiological significance.

Introduction

Activation of G-protein coupled receptors (GPCRs) by extracellular stimuli such as neurotransmitters, hormones or light induces an intracellular signal cascade mediated by heterotrimeric GTP-binding proteins (G-proteins). GPCRs are a large receptor family with a similar structure of seven transmembrane (TM) domains which exhibit highly conserved amino acid sequences (Gether and Kobilka, 1998, Strader et al., 1995). Consequently, this high amount of amino acid conservation among the GPCR family members has enabled the discovery of novel, unknown GPCRs from sequence information alone without resorting to pharmacological techniques. Although most of these novel GPCRs are so-called orphan GPCRs, for which the endogenous ligands have not been identified, GPCRs for classical neurotransmitters and hormones have proven to be such excellent therapeutic targets that orphan GPCRs are the focus of much interest (Stadel et al., 1997).

Recent efforts to sequence the human genome have focused on the random sequencing of cDNAs, resulting in the characterization of many novel expressed sequence tags (ESTs). Once characterized, the sequences of these ESTs are stored in databases, accelerating discovery of new GPCRs in silico. However, many genes, particularly those expressed in low abundance, have yet to be identified in these EST databases due to the non-targeted, random nature of the sequencing. Hence, in order to identify novel GPCRs expressed at low levels in tissues, low stringency hybridization and degenerate PCR screening is still useful.

During a previous study (Matsumoto et al., 1996a, Matsumoto et al., 1996b), a cDNA probe for the dopamine D4 receptor (Van Tol et al., 1991) was found to cross-hybridize at low stringency with mRNAs other than the D4 mRNA in human brain regions, including the cerebral cortex. This result indicated both the possible existence of other GPCRs which share homology with the D4 receptor and the feasibility of cloning those GPCR genes by using low stringency screening with the D4 probe. Therefore, a human cerebral cortex cDNA library was screened using the D4 probe at low stringency to discover other novel GPCRs.

Section snippets

Low stringency screening of a human cDNA library

A human cerebral cortex cDNA library was purchased from Clontech Labs. Approximately 1×10⁶ plaques were transferred to nylon membranes and hybridization was done with a probe corresponding to the human D4 cDNA containing TM domains VI and VII (position 1138–1406; referred to the HUMD4C sequence in the GenBank database). The membranes were hybridized with this ³²P-labeled probe (1×10⁶ cpm/ml) for 20 h at 30°C in buffer containing 5×SSPE, 5×Denhardt's, 0.1% SDS, 100 μg/ml denatured salmon sperm DNA

Screening of the human cerebral cortex cDNA library

A human cerebral cortex cDNA library was screened at low stringency using a cDNA probe derived from the region of the dopamine D4 receptor TM VI–VII. 17 positive clones were isolated; sequence analysis revealed these were all independent clones. All these clones except two, which encoded spectrin, were found to encode GPCRs. As shown in Table 1, the clones obtained were mostly previously known GPCRs for noradrenaline, acetylcholine, 5-hydroxytryptamine, and the neuropeptide orphanin

Discussion

A low-stringency survey for novel GPCRs in a human cerebral cortex cDNA library, using a dopamine D4 receptor probe, identified cDNA clones encoding the novel GPCR SALPR and the human ortholog for rat H218, as well as cDNA clones of known GPCRs for noradrenaline, acetylcholine, 5-hydroxytryptamine and orphanin FQ/nociceptin. The variation in type of clones obtained was due in part to the nature of the D4 probe used; its high GC content and the high level of sequence conservation in TM VI–VII

Acknowledgements

We thank Dr Shigekazu Nagata for providing pEF-BOS, Mr Steven E. Johnson for help in preparing the manuscript and Ms Kayo Aiki for technical assistance.

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The novel G-protein coupled receptor SALPR shares sequence similarity with somatostatin and angiotensin receptors

Abstract

Introduction

Section snippets

Low stringency screening of a human cDNA library

Screening of the human cerebral cortex cDNA library

Discussion

Acknowledgements

FEBS Lett.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Mol. Cell. Neurosci.

Mol. Brain Res.

Neurosci. Lett.

J. Biol. Chem.

FEBS Lett.