title: Characterization of a PTH1R missense mutation responsible for Jansen type metaphyseal chondrodysplasia.
abstract: Parathyroid hormone and parathyroid hormone-related peptide (PTHrP), and its receptor (PTH1R) play an important role in differentiation of bone and cartilage in the developing stages. Constitutive dimers of PTH1R are believed to be dissociated by ligand binding, and monomeric PTH1R is capable of activating G protein. Jansen type metaphyseal chondrodysplasia is caused by missense mutations in PTH1R, which are constitutively active even without the presence of the ligands. However, the underlying pathomechanisms remained largely unknown. In this study, we have attempted to further characterize a PTH1R missense mutation H223R responsible for Jansen type metaphyseal chondrodysplasia. cDNAs encoding wild-type (Wt)- and H223R mutant (Mut)-PTH1R were transfected into HEK293T cells, and as a consequence of western blots, both the Wt- and Mut-PTH1R proteins showed several fragments between 55 and 65 kDa in size, while the patterns of N-glycosylation were distinct between them. Then we hypothesized that the Mut-PTH1R might physically interact with the Wt-PTH1R, leading to affect the downstream cAMP accumulation. Co-immunoprecipitation assays clearly showed that interaction occurred not only between the Wt-PTH1R themselves, but also between the Wt- and Mut-PTH1R. Furthermore, we performed CRE reporter assays to investigate cAMP accumulation. Constitutive, ligand-independent cAMP accumulation was observed in HEK293T cells expressing the Mut-PTH1R. Interestingly, there was a statistically lower constitutive activity in HEK293T cells co-expressing the Wt- and Mut-PTH1R proteins. Summarizing, it seems likely that Mut-PTH1R may be, at least in part, co-localized with Wt-PTH1R by forming a heterodimer, leading to affect the function to each other.
authors.affiliation: Department of Pediatric Dentistry, The Nippon Dental University School of Life Dentistry at Niigata, 1-8 Hamauracho, Chuo-ku, Niigata, 951-8580, Japan. jshimo@ngt.ndu.ac.jp.
authors.forename: Junko
authors.initials: J
authors.lastname: Shimomura-Kuroki
authors.affiliation: Laboratory of Genetic Skin Diseases, Niigata University Graduate School of Medical and Dental Sciences, 1-757, Asahimachi-dori, Chuo-ku, Niigata, 951-8510, Japan.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Farooq
authors.affiliation: Department of Pediatric Dentistry, The Nippon Dental University School of Life Dentistry at Niigata, 1-8 Hamauracho, Chuo-ku, Niigata, 951-8580, Japan.
authors.forename: Tsuneo
authors.initials: T
authors.lastname: Sekimoto
authors.affiliation: Department of Developmental Biology of Hard Tissue, Hokkaido University Graduate School of Dental Medicine, Kita 13 Nishi 7, Kita-ku, Sapporo, 060-8586, Japan.
authors.forename: Norio
authors.initials: N
authors.lastname: Amizuka
authors.affiliation: Department of Dermatology, Niigata University Graduate School of Medical and Dental Sciences, 1-757, Asahimachi-dori, Chuo-ku, Niigata, 951-8510, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Shimomura
mesh_terms: D015153:Blotting, Western; D000242:Cyclic AMP / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D020125:Mutation, Missense; D010009:Osteochondrodysplasias / Q000235:genetics*; D016133:Polymerase Chain Reaction; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000235:genetics*; D014162:Transfection
chemical_list: C473449:PTH1R protein, human; D044168:Receptor, Parathyroid Hormone, Type 1; D000242:Cyclic AMP
keywords: Dimerization; G protein-coupled receptor; Jansen type metaphyseal chondrodysplasia; PTH; PTH1R