Interaction of the calcium-sensing receptor and filamin, a potential scaffolding protein

In many cases, the biologic responses of cells to extracellular signals and the specificity of the responses cannot be explained solely on the basis o f the interactions of known signaling proteins. Recently, scaffolding and a daptor proteins have been identified that organize signaling proteins in ce lls and that contribute to the nature and specificity of signaling pathways . In an effort to identify proteins that might organize the signaling syste m(s) activated by the extracellular Ca2+ receptor (CaR), we used a bait con struct representing the intracellular C terminus of the human Call and the yeast two hybrid system to screen a human kidney cDNA library. We identifie d a clone representing the C-terminal 1042 amino acids (aa) of the cytoskel etal protein filamin (ABP-280). Analysis of truncation and deletion constru cts of the CaR C terminus and the filamin cDNA clone demonstrated that the CaR and filamin interact via regions containing aa 907-997 of the CaR C ter minus and aa 1566-1875 of filamin. Interaction of the two proteins in mamma lian REK-293 cells was demonstrated by co-immunoprecipitation and colocaliz ation of them using immunofluorescence microscopy. The functional importanc e of their interaction was documented by transiently expressing the CaR in M2 melanoma cells that lack filamin, or in A7 melanoma cells that stably ex press filamin, and demonstrating that the CaR activated ERK only in the pre sence of filamin. Co-expression of the CaR with a peptide derived from the region of the CaR C terminus that interacts with filamin reduced the abilit y of the CaR to activate p42ERK in a dose-dependent manner, but did not inh ibit the ability of the ETA receptor to activate ERK. The fact that filamin interacts with the CaR and other cell signaling proteins including mitogen -activated protein kinases and small GTPases, indicates that it may act as a scaffolding protein to organize cell signaling systems involving the CaR.