title: Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling.
abstract: The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.
authors.affiliation: Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009, Australia.
authors.forename: Aaron L
authors.initials: AL
authors.lastname: Magno
authors.forename: Evan
authors.initials: E
authors.lastname: Ingley
authors.forename: Suzanne J
authors.initials: SJ
authors.lastname: Brown
authors.forename: Arthur D
authors.initials: AD
authors.lastname: Conigrave
authors.forename: Thomas
authors.initials: T
authors.lastname: Ratajczak
authors.forename: Bryan K
authors.initials: BK
authors.lastname: Ward
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D060588:LIM Domain Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins; D044169:Receptors, Calcium-Sensing / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: D003598:Cytoskeletal Proteins; D060588:LIM Domain Proteins; D016601:RNA-Binding Proteins; D044169:Receptors, Calcium-Sensing; C429688:TES protein, human; D054460:rho-Associated Kinases