Title

Kalirin-12 coordination of cytoskeletal changes and endocytosis

Date of Completion

January 2005

Keywords

Biology, Molecular|Biology, Neuroscience|Biology, Cell

Degree

Ph.D.

Abstract

Guanine nucleotide exchange factors (GEFs) and their target Rho GTPases are key regulators of cytoskeletal and membrane trafficking in neuronal growth cones. Kalirin, a neuronal Rho-GEF, plays an essential role in maintenance of spines and the dendritic tree. Early in development, alternative splicing of the Kalirin gene yields Kalirin-12, an isoform that includes immunoglobulin (Ig), fibronectinIII (FnIII), and kinase domains. Immunocytochemical and biochemical approaches identified Kalirin-12 in embryonic growth cones. Binding assays utilizing GST-Kalirin-IgFnIII identified dynamin, a GTPase essential for clathrin-mediated endocytosis, and GRIP1, a scaffolding protein that binds the GluR2 subunit of the AMPA receptor, as Kalirin-12 interactors. ^ Both the IgFnIII domain and full-length Kalirin-12 interact with dynamins 1 and 2. Co-immunoprecipitation experiments demonstrate an interaction of Kalirin-12 with dynamins 1 and 2 and with GRIP in embryonic brain. Dynamin 2 is enriched in embryonic growth cones and co-immunoprecipitates with Kalirin-12 from solubilized growth cone membranes. Over-expression of Kalirin-12 disrupts clathrin-mediated endocytosis in non-neuronal cells, PC12 cells and striatal neurons. An essential role for Kalirin in clathrin-mediated endocytosis is suggested by the impaired endocytosis that occurs following expression of a Kalirin-12 antisense vector in PC12 cells. Co-immunoprecipitation demonstrates an interaction of Kalirin-12 with clathrin, AP180, cortactin and α-adaptin. Its ability to interact with multiple proteins may allow Kalirin-12 to coordinate Rho GTPase-mediated changes in the actin cytoskeleton with dynamin-mediated changes in membrane trafficking. ^ Over-expression studies suggest that both the IgFnIII and kinase domains of Kalirin play roles in endocytosis. When over-expressed in primary striatal neurons, both the active kinase domain and the inactive mutant kinase domain of Kalirin induce process outgrowth. Over-expression of the inactive kinase increases neuronal branching and total neurite length. Thus the kinase domain of Kalirin regulates process outgrowth as well as clathrin-mediated endocytosis. ^ In the adult brain, Kalirin-12 and dynamin are present in the post-synaptic density. The interactions mediated by its IgFnIII domain and defined in the embryonic brain may govern the function of Kalirin-12 in the adult brain. Instead of promoting process outgrowth, these interactions may allow Kalirin-12 to play a role in synaptogenesis and in modifying neuronal structure in response to varying inputs. ^