Direct interaction of the 170 kDa isoform of synaptojanin 1 with clathrin and with the clathrin adaptor AP-2

Synaptojanin 1, a polyphosphoinositide phosphatase, is expressed as two maj or alternatively spliced isoforms of 145 kDa (SJ145) and 170 kDa (SJ170) [1 ,2], which are thought to have pleiotropic roles in endocytosis, signaling and actin function [3-5]. SJ145 is highly enriched in nerve terminals where it participates in clathrin-dependent synaptic vesicle recycling [1,5]. SJ 170, which differs from SJ145 by the presence of a carboxy-terminal extensi on, is the predominant isoform in developing neurons and is expressed in a variety of tissues [2]. The carboxy-terminal domain unique to SJ170 was pre viously shown to bind Eps15 [6], a protein involved in receptor-mediated en docytosis. Here, we show that the same domain also binds clathrin and the c lathrin adaptor AP-2. These interactions occur both in vitro and in vivo an d are direct. Binding of AP-2 is mediated by the ear domain of its alpha-ad aptin subunit and binding of clathrin by the amino-terminal domain of its h eavy chain. Overexpression in chinese hamster ovary (CHO) cells of full-len gth SJ170 or its unique carboxy-terminal region caused mislocalization of E ps15, AP-2 and clathrin, as well as inhibition of clathrin-dependent transf errin uptake. These findings suggest a close association of SJ170 with the clathrin coat and provide new evidence for its physiological role in the re gulation of clathrin coat dynamics.