Generation of a constitutively active mutant of human GPR48/LGR4, a G-protein-coupled receptor.

GPR48, also known as leucine-rich repeat (LRR)-containing G-protein-coupled receptor 4 (LGR4), is a member of the G-protein-coupled receptor (GPCR) family of proteins. However, its biological functions remain unclear, since neither its ligand nor signal transduction pathway have been identified, and it is usually difficult to solve the function of such orphan receptors. The aim of this study was to generate a constitutively active form of human GPR48, that would form a ligand-independent active conformation and may function in a similar manner to activated GPR48 following ligand binding. We introduced four independent mutations into transmembrane domains V and VI of a human GPR48 cDNA. The wild-type and mutant GPR48s were expressed in HEK293 cells by transient transfection of appropriate expression plasmids. Since ligand-activated receptors for gonadotropins, which are structurally similar to GPR48, stimulate adenylate cyclase and increase cellular cyclic AMP, we investigated, whether the GPR48-transfected cells showed altered cyclic AMP levels. The cellular cyclic AMP level in HEK293 cells was increased following transfection of wild-type GPR48 in a dose-dependent manner. Moreover, transfection of a GPR48-T7551 mutant, in which threonine-755 was replaced with isoleucine, dramatically increased the cyclic AMP level. Stable transformants derived from HCT116 cells that constitutively expressed the GPR48-T7551 mutant also showed high cyclic AMP levels. These results indicate that the GPR48-T7551 mutant is a constitutively active mutant. This mutant may be useful for studying the biological functions of GPR48 and GPR48-mediated signal transduction, even if the specific ligand remains unknown in the future.