Huntingtin interacting protein 1 Is a clathrin coat binding protein required for differentiation of late spermatogenic progenitors

Abstract

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 beta 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.

FIG. 1

Nucleotide sequence of the HIP1 promoter, the newly identified exon 1, and domain structure of the HIP1 protein. (a) Promoter and coding sequence of HIP1 exon 1. Promoter sequences are shown, with the CCAAT box underlined and bold and GC boxes in bold. Putative initiator methionines are shown in bold. The first of these contains the strongest Kozak consensus sequence (15). Only the italicized sequence was previously available through the National Center for Biotechnology Information. (b) HIP1 protein domains. The amino acid sequence of HIP1 exhibits homology to previously characterized domains in other proteins, as noted in the figure. Abbreviations: ENTH, epsin N-terminal homology; Clathrin, putative clathrin binding site; AP-2, putative AP-2 binding site; LZ, leucine zipper. The asterisk shows the breakpoint in the HIP1-PDGFβR fusion protein (23).

FIG. 2

Immunofluorescence confocal analysis of the intracellular distribution of endogenous hHIP1 (green) in 293T cells and its localization with respect to Eps15 (red). (a) HIP1 is localized to punctate vesicle-like structures. Labeling of HIP1 was done with anti-HIP1 monoclonal antibody 4B10 and anti-mouse FITC secondary antibody. The staining pattern with anti-HIP1 monoclonal antibodies is punctate, and the spots are distributed evenly in the cytoplasm. A similar pattern was obtained using anti-HIP1 polyclonal antibody. All subsequent HIP1 labeling was done with the monoclonal antibodies since there is little background and no signal in the red spectrum. (b) Double labeling of HIP1 (green) and eps15 (red) and overlay of the two (bottom right panel). The differential interference contrast/Nomarski images (bottom left panel) highlight cell morphology and identify the nuclei.

FIG. 3

HIP1 associates with clathrin. (a) Immunologic analysis of 293T cell extracts prepared using various lysis buffers. Lanes: 1, extract prepared with lysis buffer A, immunoprecipitating (IP) antibody preimmune rabbit serum; 2, extract prepared with lysis buffer B; 3, extract prepared with lysis buffer C; 4, extract prepared with lysis buffer A, immuniprecipitating antibody anti-3′hHIP1 polyclonal antibody; 5, extract prepared with lysis buffer B; 6, extract prepared with lysis buffer C. Lysis buffer A is defined in Materials and methods. Lysis buffer B is the same as buffer A but with 1.5 mM MgCl₂, 5 mM EGTA, and 10% glycerol added; lysis buffer C is the same as lysis buffer B without NaCl. (b) Immunologic analysis of A549 lung cancer cell lines for the HIP1 association with clathrin. The immunoprecipitating antibody was the same as in lanes 4 to 6 of panel A. In both panels, the immunoprecipitated proteins were Western blotted (WB) with either anti-clathrin or anti-HIP1 antibodies, as indicated.

FIG. 4

Targeted disruption of murine HIP1 by homologous recombination. (a) Targeted deletion of a 13.7-kb segment of the murine HIP1 gene, including exons 2 to 7. The targeting vector contained a loxP flanked Neo^r cassette. Homologous recombination between the 5′ and 3′ sequences flanking the knockout region results in a recombinant allele with a loxP-flanked Neo^r in the same orientation as HIP1. If a transcript were spliced between exons 1 and 8, translation would result in a premature stop codon. Abbreviations: Ap, ApaI; B, BamHI; E, EcoRI; H, HindIII; K, KpnI; Sph, SphI; X, XbaI. (b) Southern blot analysis of genomic DNA from tail biopsy specimens screened with the 5′ probe. Screening of genomic DNA digested with EcoRI with 5′ probe results in 16.5-kb wild-type (WT) and 12.0-kb recombinant (rec) bands. Abbreviations: +/+, wild type; +/−, heterozygote; −/−, homozygote. (c) Western blot analysis of brain and testicular extracts from HIP1^+/+ and HIP1^−/− mice. The 111- and 194-kDa marker bands are indicated. The HIP1 band was absent in extracts from HIP1^−/− mice. (d) Northern blot analysis of normal fetal mRNA for wild-type HIP1. The blot was purchased from Clontech and probed with a ³²P-labeled mHIP1 cDNA fragment carrying nucleotides 1 to 1599.

FIG. 5

Analysis of testes from HIP1 mutant mice. (a to c) Photomicrographs of testicular sections show seminiferous tubules from 6-week-old +/+, +/−, and −/− animals, stained with hematoxylin and eosin. Arrows in panels b and c show multinucleated giant cells not seen in the testes of +/+ animals. Magnification, ×100. (d and e) High-power photomicrographs of multinucleated giant cells, stained with hematoxylin and eosin. Magnification, ×1,000 under oil immersion. (f to h) Photomicrographs of epididymides from the same animals in panels a to c. There were fewer mature sperm in the epidiymides of the −/− animal (h). Magnification ×400. (i) Quantitative analysis of the number of giant cells seen on each section (shown as mean and standard error of the mean). Testicular sections (n = 10 to 16 for each genotype) were scored for the number of multinucleated giant cells seen. For +/+ mice, the range was 0 to 2 giant cells/section, while for −/− mice, the number ranged from 2 to 28 giant cells/section. ∗, P < 0.005 for comparison between +/+ and −/− mice.

FIG. 6

Increased apoptosis in HIP1^−/− mouse testes without reductions in the spermatogonial cell population. (a to c) Photomicrographs of TUNEL assays in 6-week-old +/+, +/−, and −/− mice, stained with NBT-BCIP and counterstained with Nuclear Fast Red. In the +/+ mice, a few deep violet-black cells were seen (a), but note the large numbers of TUNEL-positive cells seen in the −/− section (c). +/− sections showed a large variability in the number of TUNEL-positive cells (b). All testes were fixed in 10% formalin in PBS for this purpose. Magnification, ×89. (d to f) Immunohistochemical analysis for GCNA. GCNA-positive cells are stained dark brown. No counterstain was used in these sections. There was no difference in the number of GCNA-positive cells among +/+, +/−, and −/− mice. Magnification, ×89. (g) Number of TUNEL-positive cells per section, averaged by genotype (mean and standard error of the mean). Six testicular sections of each genotype were subjected to TUNEL assay, and dark violet-black cells were counted on each section. ∗, P<0.001 for comparison between +/+ and −/− mice.

FIG. 7

HIP1 is expressed only in the postmeiotic spermatids of the seminiferous tubules. A monoclonal antibody was generated against a human HIP1 recombinant GST fusion protein and designated HIP1 MAb4B10. Human tissue was fixed in formaldehyde, and standard methods were used to stain with the HIP1 antibody, MAb4B10. (a) Low-power view of the seminiferous tubules showing positive staining (brown) in the center of the tubules. (b to d) Higher-power views (×74, ×296, and ×740, respectively). demonstrating that only the round postmeiotic spermatids express HIP1. Spermatogonia, primary and secondary spermatocytes, and Sertoli cells do not show expression of HIP1.