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First published online April 3, 2008
doi: 10.1242/10.1242/jcs.025833

Journal of Cell Science 121, 1264-1274 (2008)
Published by The Company of Biologists 2008
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Internalization of LDL-receptor superfamily yolk-protein receptors during mosquito oogenesis involves transcriptional regulation of PTB-domain adaptors

Sanjay K. Mishra1,*, Anupma Jha1,*, Amie L. Steinhauser1, Vladimir A. Kokoza2, Charles H. Washabaugh1, Alexander S. Raikhel2, Woodbridge A. Foster3 and Linton M. Traub1,{ddagger}

1 Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
2 Department of Entomology, University of California, Riverside, CA 92521, USA
3 Department of Entomology, Ohio State University, Columbus, OH 43210, USA


Figure 1
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  		Fig. 1. Mosquito ARH-like orthologues. (A) Schematic of the domain organization of human (Hs), Xenopus laevis (Xl), A. gambiae (Ag) and A. aegypti (Aa) ARH and ARH-like proteins. The location of clathrin- (red) and AP-2 β2- (green) or putative {alpha}-appendage-binding (blue) interaction motifs is shown. (B) Sequence alignment of the PTB-domain regions of D. melanogaster (Dm) Numb (FlyBase Gene ID CG3779; PBD accession number: 1DDM), human ARH and Anopheles and Aedes ARH-like proteins. Numbered secondary structure elements ({alpha} helix, blue; β-strand, green) of the Numb PTB domain are indicated above. Identical residues (magenta) and chemically conservative substitutions (yellow) are boxed. Positionally conserved basic residues that, based on the structures of the Dab1 and Dab2 PTB domains, are probably involved in phosphoinositide binding (vertical arrowheads) and in FXNPXY-sequence engagement (vertical arrows) are indicated. (C) Sequence alignment of the C-terminal portions of the Anopheles, Aedes and Tribolium castaneum (Tc) ARH-like proteins. Conservation colored as in B and the location of the putative clathrin (red) and AP-2 {alpha}-appendage (blue) interaction motifs is indicated.

 

Figure 2
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  		Fig. 2. Phosphoinositide binding of the A. aegypti ARH-like protein. (A) Human GST-ARH (60 µg/ml, residues 1-308; lanes a-d), GST-AaARH (residues 21-292; lanes e-h) or GST-AaARH PTB domain (residues 21-192, lanes i,j,k,l) were incubated with synthetic liposomes without (lanes a,b,e,f,i,j) or with PtdIns(4,5)P2 (lanes c,d,g,h,k,l) on ice in the presence of 100 µg/ml BSA. After centrifugation, aliquots of 3% of each supernatant (S) and 25% of each pellet (P) were resolved by SDS-PAGE and stained with Coomassie Blue. The position of the molecular mass standards (in kDa) is indicated. (B) GST-AaARH (60 µg/ml, residues 21-292, lanes a-f) or GST-ARH (residues 1-308, lanes g,h) were incubated with PtdIns(4,5)P2 liposomes in presence of 100 µg/ml BSA and either no (lanes a and b) or increasing concentrations (3 µg, lanes c,d or 6 µg, lanes e-j) of clathrin. After centrifugation, aliquots of 3% of each supernatant (S) and 25% of pellets (P) were resolved by SDS-PAGE and stained. The position of the clathrin heavy chain (HC) is indicated.

 

Figure 3
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  		Fig. 3. Protein interactions in a yeast two-hybrid assay. Saccharomyces cerevisiae strain AH109 were transformed with the indicated plasmid combinations containing the pGBKT7-binding domain (BD) and the pGADT7-activation domain (AD), and spotted onto SD minimal medium plates lacking either Leu and Trp or His, Leu and Trp supplemented with 0.75 mM 3-amino-1,2,4-triazole (3-AT) and grown at 30°C.

 

Figure 4
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  		Fig. 4. Coat-binding properties of mosquito ARH-like proteins. (A) GST (100 µg, lanes a and b) or GST-AaARH (residues 21-292; lanes c and d) or human GST-ARH (residues 1-308; lanes e and f) on glutathione-Sepharose was incubated with rat brain cytosol. After centrifugation, aliquots of 2% of each supernatant (S) and 12.5% of each washed pellet (P) were resolved by SDS-PAGE and stained or transferred to nitrocellulose. Portions of the blots were probed with the anti-clathrin heavy chain (HC) mAb TD.1 and AP-1/2 β subunit antibody GD/1, or anti-µ2 subunit serum, or anti-{alpha} subunit mAb clone 8, or anti-clathrin light chain (LC) mAb Cl57.3. Only the relevant portion of each blot is shown. (B) GST (~250 µg, lanes a and b), GST-ARHC1 (lanes c and d), GST-AgARHC1 (residues 196-300; lanes e and f), GST-AgARHC1 (LID->AAA) (lanes g and h), or AgARHC1 (W268A) (lanes i and j) on glutathione-Sepharose was incubated with rat brain cytosol. After centrifugation, aliquots of 2.5% of each supernatant (S) and 12.5% of washed pellets (P) were resolved by SDS-PAGE and stained or transferred to nitrocellulose. Portions of the blots were probed with the anti-clathrin HC mAb TD.1 and affinity purified anti-AP-1/2 β subunit antibody GD/1, or anti-µ2 subunit serum or affinity purified anti-µ1 antibody RY/1. Note that only the human ARHC1-fusion protein binds to the µ1 subunit (AP-1) as a consequence of the selectivity for the β-subunit appendage. The mammalian AP-1 β1 and AP-2 β2 subunits are ~85% identical and the appendages share binding partners.

 

Figure 5
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  		Fig. 5. Appendage selectivity of the mosquito ARH-like proteins. (A) GST (~400 µg, lanes a,b,e,f) or GST-AgARHC1 (residues 196-300; lanes c,d,g,h) immobilized on glutathione-Sepharose was incubated with 100 µg/ml of either thrombin-cleaved {alpha}C appendage (open arrowheads) or β2 appendage (arrowheads) in the presence of 25 µM PPACK and 100 µg/ml BSA. After centrifugation, 2.5% of each supernatant (S) and 12.5% of each washed pellet (P) were resolved by SDS-PAGE for staining or fivefold less of each supernatant and pellet were transferred onto nitrocellulose. Blots were probed with either anti-AP-2 {alpha} subunit mAb 100/2 or affinity-purified rabbit anti-AP-2 β2 subunit antibodies. (B) GST (~250 µg, lanes a,b,e,f) or GST-AaARH (residues 20-292) on glutathione-Sepharose was incubated with either 50 µg/ml thrombin-cleaved {alpha}C wild type (lanes a-d) or {alpha}C (Q782A) mutant (lanes e-h) in the presence of 100 µg/ml BSA. After centrifugation, 2.5% of each supernatant (S) and 12.5% of washed pellets (P) were resolved on SDS-PAGE for staining or fivefold less of each supernatant and pellet transferred onto nitrocellulose. The blot was probed with the anti-AP-2 {alpha} subunit mAb 100/2. Stained {alpha} appendage in supernatant (open arrowheads) and pellet (arrowhead) fractions is indicated.

 

Figure 6
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  		Fig. 6. Subcellular localization of a tagged AgARH-like protein. HeLa cells, transiently transfected with the GFP-AgARH-like protein, were fixed and stained with mAb AP.6 directed against the {alpha} subunit of the AP-2. GFP (green, A,D,F,I) and AP-2 (red, B,D',G,J) signals were collected sequentially by confocal microscopy and representative single optical sections are shown. Scale bar: 20 µm. An enlarged region illustrating the degree of colocalization (D"; arrows) of GFP-AgARH-like fusion (D) with AP-2 (D') is shown, as well as an example of large intracellular aggregates of the fusion protein below the nucleus (F) also containing AP-2 (G,H; arrows). A region where several adjacent cells mechanically detached from the glass surface leaving only adherent ventral plasma membrane (E) shows a high degree of colocalization of the GFP-AgARH-like protein (yellow) compared with the non-transfected cells (red). The generally diffuse intracellular distribution of the doubly mutated GFP-AgARH-like (LID->AAA/W268A) mutant is shown (panels I-K).

 

Figure 7
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  		Fig. 7. mRNA expression in Aedes ovaries. RT-PCR using RNA isolated from either previtellogenic (lanes b,e,h) or 16 hour post-blood-meal ovaries or with RNA derived from cultured Aag2 cells and primer pairs designed to amplify a 0.81 kb AaARH, 0.56 kb vitellogenin receptor (VgR) or a 0.5 kb AP-2 {alpha} subunit product. As controls, PCR with the identical primer sets and DNA plasmid template (lanes a,d,g) was performed. The position of the DNA standards (in kb) is indicated.

 

Figure 8
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  		Fig. 8. Changes in the ARH-like transcript during A. aegypti egg chamber development. Whole-mount in situ hybridization analysis of ovaries dissected from previtellogenic (A), 6 hour (B) or 20 hour post-blood-meal (C-F) females. Fixed ovaries were hybridized with antisense AaARH-like (A-D), vitellogenin receptor (VgR; E) or no (F) riboprobe. The purple staining indicates the positioning of mRNA transcripts. Samples in A-D were treated identically and the images obtained under the same conditions. fc, follicle cells; g, germarium; nc, nurse cells; o, oocyte; sf, secondary follicle.

 

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© The Company of Biologists Ltd 2008