HIP1 and HIP1r Stabilize Receptor Tyrosine Kinases and Bind 3-Phosphoinositides via Epsin N-terminal Homology Domains

Abstract

Huntingtin-interacting protein 1-related (HIP1r) is the only known mammalian relative of huntingtin-interacting protein 1 (HIP1), a protein that transforms fibroblasts via undefined mechanisms. Here we demonstrate that both HIP1r and HIP1 bind inositol lipids via their epsin N-terminal homology (ENTH) domains. In contrast to other ENTH domain-containing proteins, lipid binding is preferential to the 3-phosphate-containing inositol lipids, phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,5-bisphosphate. Furthermore, the HIP1r ENTH domain, like that of HIP1, is necessary for lipid binding, and expression of an ENTH domaindeletion mutant, HIP1r/⌬E, induces apoptosis. Consistent with the ability of HIP1r and HIP1 to affect cell survival, full-length HIP1 and HIP1r stabilize pools of growth factor receptors by prolonging their half-life following ligand-induced endocytosis. Although HIP1r and HIP1 display only a partially overlapping pattern of protein interactions, these data suggest that both proteins share a functional homology by binding 3-phosphorylated inositol lipids and stabilizing receptor tyrosine kinases in a fashion that may contribute to their ability to alter cell growth and survival.Huntingtin-interacting protein 1-related (HIP1r) 1 is a clathrin-binding protein that is the only known mammalian relative of huntingtin-interacting protein 1 (HIP1) (1), a protein that transforms fibroblasts via undefined mechanisms (2). HIP1 is also part of a t(5;7) chromosomal translocation that results in expression of an oncogenic HIP1/PDGF␤R fusion protein that causes myelomonocytic leukemia (3). Furthermore, HIP1 is overexpressed in multiple primary epithelial tumors, and overexpression in prostate tumors predicts progression of prostate cancer (4). The transformation of fibroblasts by HIP1 is associated with altered levels of growth factor receptors (2).The mechanism by which HIP1 overexpression alters growth factor receptor levels may be a result of its role in trafficking of growth factor receptors. HIP1 and HIP1r each contain a clathrin light chain-binding coiled-coil region (5), a leucine zipper, and a C-terminal TALIN homology domain. TALIN is a protein that binds actin and is involved in cell-substratum interactions (6). In addition, HIP1 and HIP1r contain epsin N-terminal homology (ENTH) domains. This domain in epsin and AP180 predominantly binds to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) and is important in regulating clathrin-mediated endocytosis (7, 8). HIP1 and HIP1r both have a punctate immunolocalization and co-localize partially with clathrin, AP-2, and endocytosed transferrin (9 -13).Thus, structural and functional data suggest that HIP1 and HIP1r are involved in vesicle trafficking either at the plasma membrane, during sorting of vesicles, or at the trans-Golgi network. Unlike HIP1, HIP1r has lower affinity for clathrin, does not bind ␣-adaptin (5), does not bind huntingtin directly, and does bind actin via its TALIN homology domain (9). HIP1 homologues have been found...

Use of a Cryptic Splice Site for the Expression of Huntingtin Interacting Protein 1 in Select Normal and Neoplastic Tissues

“…Interestingly, these truncated fusion proteins displayed a higher specific binding to PtdIns(3)P than to polyphosphorylated inositol lipids. On the other hand, our previous studies using the full-length HIP1 expressed in 293T cells indicated that its lipid-binding preference was polyphosphorylated 3-phosphoinositides (29). Our contradictory results may be due to either the absence of the carboxyl terminal end of the protein, differences in proteins expressed in bacteria versus mammalian cells, or differences in the materials used in the various assays.…”

Hip1r is expressed in gastric parietal cells and is required for tubulovesicle formation and cell survival in mice

“…The changes in cell morphology in the Hip1r knockdown cell studies suggested that Hip1r plays a global role in regulating actin and membrane dynamics. Furthermore, overexpression of both Hip1 and Hip1r has previously been shown to stabilize growth factor receptors on the cell surface (9). This is the putative mechanism of transformation by Hip1 when overexpressed in certain human cancers (6).…”

Novel Function of Clathrin Light Chain in Promoting Endocytic Vesicle Formation

“…Therefore, it is hypothesized that Hip1/R promote clathrin assembly by releasing LC's inhibition of lattice assembly (Chen and Brodsky, 2005;Legendre-Guillemin et al, 2005). Hip1 family members also interact with phosphatidyl-inositol lipid through an N-terminal ANTH domain and F-actin through a talin-Hip1/R/Sla2p actin-tethering C-terminal homology (THATCH) domain, suggesting that these proteins also serve to coordinate the actin cytoskeleton at clathrin-coated pits (McCann and Craig, 1997;Engqvist-Goldstein et al, 1999;Legendre-Guillemin et al, 2002;Hyun et al, 2004;Senetar et al, 2004;Sun et al, 2005).…”