A megalin-like receptor is involved in protein endocytosis in the midgut of an insect (Bombyx mori, Lepidoptera).
The mechanism responsible for fluorescein isothiocyanate (FITC)-albumin internalization by columnar cells in culture obtained from the midgut of Bombyx mori larvae was examined by confocal laser scanning microscopy. Protein uptake changed over time, and it appeared to be energy dependent, since it was strongly reduced by both low temperatures and metabolic inhibitors. Labeled albumin uptake as a function of increasing protein concentration showed a saturation kinetics with a Michaelis constant value of 2.0 +/- 0.6 microM. These data are compatible with the occurrence of receptor-mediated endocytosis. RT-PCR analysis and colocalization experiments with an anti-megalin primary antibody indicated that the receptor involved was a putative homolog of megalin, the multiligand endocytic receptor belonging to the low-density lipoprotein receptor family, responsible for the uptake of various molecules, albumin included, in many epithelial cells of mammals. This insect receptor, like the mammalian counterpart, required Ca(2+) for albumin internalization and was inhibited by gentamicin. FITC-albumin internalization was clathrin mediated, since two inhibitors of this process caused a significant reduction of the uptake, and clathrin and albumin colocalized in the intermicrovillar areas of the apical plasma membrane. The integrity of actin and microtubule organization was essential for the correct functioning of the endocytic machinery.
Casartelli
M
M
Dipartimento di Biologia, Università di Milano, via Celoria 26, 20133 Milano, Italy. morena.casartelli@unimi.it
Cermenati
G
G
Rodighiero
S
S
Pennacchio
F
F
Giordana
B
B
D019297:2,4-Dinitrophenol / Q000494:pharmacology; D008841:Actin Cytoskeleton / Q000187:drug effects; D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D012831:Bombyx / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D002746:Chlorpromazine / Q000494:pharmacology; D002966:Clathrin / Q000378:metabolism; D003080:Cold Temperature; D003571:Cytochalasin B / Q000494:pharmacology; D004044:Dietary Proteins / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000378:metabolism; D041981:Gastrointestinal Tract / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D005839:Gentamicins / Q000494:pharmacology; D019476:Insect Proteins / Q000378:metabolism*; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008870:Microtubules / Q000187:drug effects; D015739:Nocodazole / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D012709:Serum Albumin / Q000378:metabolism; D019810:Sodium Azide / Q000494:pharmacology; D013696:Temperature
D001152:Arsenicals; D002966:Clathrin; D004044:Dietary Proteins; C518288:FITC-albumin; D005839:Gentamicins; D019476:Insect Proteins; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D012709:Serum Albumin; D003571:Cytochalasin B; D019810:Sodium Azide; D004492:Edetic Acid; D016650:Fluorescein-5-isothiocyanate; D019297:2,4-Dinitrophenol; D015739:Nocodazole; D002746:Chlorpromazine