The KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system

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Abstract

GPR54 is a G-protein-coupled receptor that displays a high percentage of identity in the transmembrane domains with the galanin receptors. The ligand for GPR54 has been identified as a peptide derived from the KiSS-1 gene. KiSS-1 has been shown to have anti-metastatic effects, suggesting that KiSS-1 or its receptor represents a potential therapeutic target. To further our understanding of the physiological function of this receptor, we have generated a mutant mouse line with a targeted disruption of the GPR54 receptor (GPR54 −/−). The analysis of the GPR54 mutant mice revealed developmental abnormalities of both male and female genitalia and histopathological changes in tissues which normally contain sexually dimorphic features. These data suggest a role for GPR54/KiSS-1 in normal sexual development, and indicate that study of the GPR54 mutant mice may provide valuable insights into human reproductive syndromes.

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Materials and methods

Generation of GPR54 knockout mice. Mutant mice with targeted disruption of the GPR54 gene (GPR54 −/−) were custom generated by Deltagen (Palo Alto, CA). A colony was then established at SPRI and maintained according to the guidelines and ethical standards of the SPRI ACUC.

A gene targeting vector was constructed in which a 52 bp fragment on exon 2, corresponding to position 269–320 of the ORF [5] of the GPR54 receptor gene (GenBank AF343726) was replaced with a IRES-LacZ Neo insert containing a

GPR54 −/− mouse generation and phenotypic analysis

In order to generate GPR54 −/− mice, a targeting vector was designed to disrupt a 52 bp fragment from exon 2 of the GPR54 receptor and to replace it with a IRES-LacZ Neo insert. Neo-resistant ES cells clones were then screened by Southern blot analysis of HindIII-digested genomic DNA hybridized with a radiolabeled DNA fragment that hybridizes outside of and adjacent to the construct arm (Fig. 1A). Heterozygous mice (GPR54 +/−) were interbred and homozygous mice were recovered at the expected

Discussion

It has been recently demonstrated that the decapeptide KiSS-10 (KiSS-1 residues 112–121) isolated from human placenta can activate GPR54 through the Gαq-signaling pathway [2], [4], [11]. To further our understanding of the physiological role of GPR54, we generated homozygous GPR54-deficient mice. Analysis of the phenotype of these mice revealed abnormalities in the development of both male and female homozygous mice. These abnormalities included external and internal reproductive organs which

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