Clathrin-mediated endocytosis is a major pathway for the internalization of macromolecules into the cytoplasm of eukaryotic cells. The principle coat components, clathrin and the AP-2 adaptor complex, assemble a polyhedral lattice at plasma membrane bud sites with the aid of several endocytic accessory proteins. Here, we show that huntingtin-interacting protein 1 (HIP1), a binding partner of huntingtin, copurifies with brain clathrin-coated vesicles and associates directly with both AP-2 and clathrin. The discrete interaction sequences within HIP1 that facilitate binding are analogous to motifs present in other accessory proteins, including AP180, amphiphysin, and epsin. Bound to a phosphoinositide-containing membrane surface via an epsin N-terminal homology (ENTH) domain, HIP1 associates with AP-2 to provide coincident clathrin-binding sites that together efficiently recruit clathrin to the bilayer. Our data implicate HIP1 in endocytosis, and the similar modular architecture and function of HIP1, epsin, and AP180 suggest a common role in lipid-regulated clathrin lattice biogenesis.

Huntingtin-interacting protein 1 (HIP1) is a cofactor in clathrin-mediated vesicle trafficking. It was first implicated in cancer biology as part of a chromosomal translocation in leukemia. Here we report that HIP1 is expressed in prostate and colon tumor cells, but not in corresponding benign epithelia. The relationship between HIP1 expression in primary prostate cancer and clinical outcomes was evaluated with tissue microarrays. HIP1 expression was significantly associated with prostate cancer progression and metastasis. Conversely, primary prostate cancers lacking HIP1 expression consistently showed no progression after radical prostatectomy. In addition, the expression of HIP1 was elevated in prostate tumors from the transgenic mouse model of prostate cancer (TRAMP). At the molecular level, expression of a dominant negative mutant of HIP1 led to caspase-9-dependent apoptosis, suggesting that HIP1 is a cellular survival factor. Thus, HIP1 may play a role in tumorigenesis by allowing the survival of precancerous or cancerous cells. HIP1 might accomplish this via regulation of clathrin-mediated trafficking, a fundamental cellular pathway that has not previously been associated with tumorigenesis. HIP1 represents a putative prognostic factor for prostate cancer and a potential therapy target in prostate as well as colon cancers.

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...

Huntingtin-interacting protein 1 (HIP1) interacts with huntingtin, the protein whose gene is mutated in Huntington's disease. In addition, a fusion between HIP1 and platelet-derived growth factor ␤ receptor causes chronic myelomonocytic leukemia. The HIP1 proteins, including HIP1 and HIP1-related (HIP1r), have an N-terminal polyphosphoinositide-interacting epsin N-terminal homology, domain, which is found in proteins involved in clathrin-mediated endocytosis. HIP1 and HIP1r also share a central leucine zipper and an actin binding TALIN homology domain. Here we show that HIP1, like HIP1r, colocalizes with clathrin coat components. We also show that HIP1 physically associates with clathrin and AP-2, the major components of the clathrin coat. To further understand the putative biological role(s) of HIP1, we have generated a targeted deletion of murine HIP1. HIP1 ؊/؊ mice developed into adulthood, did not develop overt neurologic symptoms in the first year of life, and had normal peripheral blood counts. However, HIP1-deficient mice exhibited testicular degeneration with increased apoptosis of postmeiotic spermatids. Postmeiotic spermatids are the only cells of the seminiferous tubules that express HIP1. These findings indicate that HIP1 is required for differentiation, proliferation, and/or survival of spermatogenic progenitors. The association of HIP1 with clathrin coats and the requirement of HIP1 for progenitor survival suggest a role for HIP1 in the regulation of endocytosis.

Sperm formation was studied in the fern, Marsilea, and the cycad, Zamia, with particular cmphasis on thc ccntrioles. In Marsilea, the maturc spcrm posscsses over 100 flagella, the basal bodies of which have the typical cylindrical structure of centriolcs. Earlier obscrvations by light microscopy suggcsted that these centrioles arise by fragmentation of a body known as the blepharoplast. In the youngest spermatids the blepharoplast is a hollow sphere approximately 0.8 tt in diameter. Its wall consists of closely packed immature ccntriolcs, or proccntriolcs. Thc procentrioles arc short cylinders which progressively lengthen during differentiation of the spermatid. At the same time thcy migrate to the surface of thc ccll, where each of thcm puts out a i]agcllum. A blepharoplast is found at cach pole of thc spindle during the last antheridial mitosis, and two blcpharoplasts are found in the cytoplasm before this mitosis. Blepharoplasts arc also found in the prcccding cell generation, but their ultimatc origin is obscure. Before thc last mitosis the blepharoplasts are solid, consisting of a clustcr of radially arranged tubulcs which bear some structural similarity to centrioles. In Zamia, similar stages are found during sperm formation, although here thc number of flagella on each sperm is close to 20,000 and thc blepharoplast measures about l0 tt in diameter. These observations arc discussed in relation to theories of ccntriolc replication.