title: Filamin-A binds to the carboxyl-terminal tail of the calcium-sensing receptor, an interaction that participates in CaR-mediated activation of mitogen-activated protein kinase.
abstract: The G protein-coupled, extracellular calcium-sensing receptor (CaR) regulates parathyroid hormone secretion and parathyroid cellular proliferation as well as the functions of diverse other cell types. The CaR resides in caveolae-plasma membrane microdomains containing receptors and associated signaling molecules that are thought to serve as cellular "message centers." An additional mechanism for coordinating cellular signaling is the presence of scaffold proteins that bind and organize components of signal transduction cascades. With the use of the yeast two-hybrid system, we identified filamin-A (an actin-cross-linking, putative scaffold protein that binds mitogen-activated protein kinase (MAPK) components activated by the CaR) as an intracellular binding partner of the CaR's carboxyl (COOH)-terminal tail. A direct interaction of the two proteins was confirmed by an in vitro binding assay. Moreover, confocal microscopy combined with two color immunofluorescence showed co-localization of the CaR and filamin-A within parathyroid cells as well as HEK-293 cells stably transfected with the CaR. Deletion mapping localized the sites of interaction between the two proteins to a stretch of 60 amino acid residues within the distal portion of the CaR's COOH-terminal tail and domains 14 and 15 in filamin-A, respectively. Finally, introducing the portion of filamin-A interacting with the CaR into CaR-transfected HEK-293 cells using protein transduction with a His-tagged, Tat-filamin-A fusion protein nearly abolished CaR-mediated activation of ERK1/2 MAPK but had no effect on ERK1/2 activity stimulated by ADP. Therefore, the binding of the CaR's COOH-terminal tail to filamin-A may contribute to its localization in caveolae, link it to the actin-based cytoskeleton, and participate in CaR-mediated activation of MAPK.
authors.affiliation: Calcium Section, Endocrine-Hypertension Division and Membrane Biology Program, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: G
authors.initials: G
authors.lastname: Hjälm
authors.forename: R J
authors.initials: RJ
authors.lastname: MacLeod
authors.forename: O
authors.initials: O
authors.lastname: Kifor
authors.forename: N
authors.initials: N
authors.lastname: Chattopadhyay
authors.forename: E M
authors.initials: EM
authors.lastname: Brown
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D002460:Cell Line; D003285:Contractile Proteins / Q000032:analysis / Q000378:metabolism*; D004789:Enzyme Activation; D064448:Filamins; D006801:Humans; D007150:Immunohistochemistry; D008840:Microfilament Proteins / Q000032:analysis / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000032:analysis / Q000502:physiology*
chemical_list: D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; D000244:Adenosine Diphosphate; D020928:Mitogen-Activated Protein Kinases