The Intersectin 2 Adaptor Links Wiskott Aldrich Syndrome Protein (WASp)-mediated Actin Polymerization to T Cell Antigen Receptor Endocytosis

Abstract

Induction of T cell antigen receptor (TCR) endocytosis has a significant impact on TCR signaling and T cell behavior, but the molecular interactions coordinating internalization of the activated TCR are poorly understood. Previously we have shown that TCR endocytosis is regulated by the Wiskott Aldrich Syndrome protein (WASp), a cytosolic effector which, upon interaction with the cdc42 Rho GTPase, couples TCR engagement to Arp 2/3 complex-mediated actin polymerization. Here we report that WASp associates in T cells with intersectin 2, an endocytic adaptor containing multiple domains including a Dbl homology (DH) domain with the potential to activate Rho GTPases. Intersectin 2 association with WASp increases after TCR engagement, and its overexpression in Cos-7 cells induces WASp translocation to endocytic vesicles within which intersectin 2 colocalizes with both WASp and cdc42. Intersectin 2, but not a DH domain-deleted (ΔDH) form of intersectin 2, and stimulation via the TCR also trigger the activation of cdc42. Induction of TCR internalization is also augmented by intersectin 2 and severely impaired by latrunculin B treatment. Thus, intersection 2 appears to function cooperatively with WASp and cdc42 to link the clathrin endocytic machinery to WASp-mediated actin polymerization and ultimately to occupancy-induced TCR endocytosis.

Intersectin 1L Guanine Nucleotide Exchange Activity Is Regulated by Adjacent src Homology 3 Domains That Are Also Involved in Endocytosis

“…Intersectin 2L participates with WASP and Cdc42 in the stimulated endocytosis of T-cell antigen receptor (TCR), which is actin dependent. The presence of the DH domain mitigated the inhibitory effects of the rest of intersectin 2L on stimulated TCR endocytosis (McGavin et al, 2001). These studies of intersectin 2L therefore support the suggestion that intersectin 1L acts to link endocytosis and actin cytoskeleton regulation.…”

“…Intersectin 1L, localized to peri-active zones Roos and Kelly, 1999), is thus well positioned to switch on actin polymerization in endocytic regions only after a burst of exocytosis. Intersectins promote actin polymerization (Hussain et al, 2001;McGavin et al, 2001) but are inhibitors of endocytosis (Sengar et al, 1999;Simpson et al, 1999;Pucharcos et al, 2000), perhaps because they sequester proteins such as dynamin that are needed for endocytosis. Because dynamin and N-WASP compete with each other for binding to the same SH3 domains of intersectin 1L, inhibition of endocytosis through sequestration of dynamin would be associated with inhibition of N-WASP binding.…”

“…In the absence of added exchange factor activity, GST RhoA, Rac1, and Cdc42 fusion proteins incorporated [ 35 S]GTP␥S to the same degree in binding studies (unpublished data). The same specificity for Cdc42 was found for intersectin 2L using a Pak-Cdc42/Rac interactivebinding domain assay (McGavin et al, 2001). We next asked whether the binding of the DH domain to small GTPases was affected by the remainder of the intersectin 1L protein.…”

“…Several recent studies address the pathways downstream of the intersectin 1L GEF activity. The intersectin 1 SH3 domains interact with not only endocytic proteins, but also with a stimulator of actin filament nucleation (Miki et al, 1996(Miki et al, , 1998Rohatgi et al, 1999), the neural Wiskott-Aldrich syndrome protein (N-WASP; Hussain et al, 2001;McGavin et al, 2001). Microinjection of intersectin 1L or the DH domain alone stimulates filopodia formation in cultured cells, an effect not seen with intersectin 1S (Hussain et al, 2001) despite its ability to interact with N-WASP.…”

Intersectin 1: a molecular linker in the central nervous system

“…In addition to endocytosis and exocytosis, intersectin 1 is also involved in actin polymerization [9,20] . Hussain et al reported that the expression of intersectin 1 DH-domain-containing constructs caused the rearrangement of actin specific for Cdc42 activation in cultured cells.…”