Identification of an insulin-responsive, slow endocytic recycling mechanism in Chinese hamster ovary cells.
In adipocytes, the insulin-regulated aminopeptidase (IRAP) is trafficked through the same insulin-regulated recycling pathway as the GLUT4 glucose transporter. We find that a chimera, containing the cytoplasmic domain of IRAP fused to transmembrane and extracellular domains of the transferrin receptor, is slowly recycled and rapidly internalized in Chinese hamster ovary cells. Morphological studies indicate that the chimera is slowly trafficked through the general endosomal recycling compartment rather than being sorted to a specialized recycling pathway. A chimera in which a di-leucine sequence within the cytoplasmic domain of IRAP has been mutated to alanines is rapidly internalized and rapidly recycled, indicating that this di-leucine is required for the slow recycling but not for the rapid internalization. Insulin stimulates a 2-3-fold increase in the recycling of the chimera and only a 1.2-fold increase in the recycling of the transferrin receptor. The effect of insulin on the recycling of the chimera is blocked by wortmannin, a phosphatidylinositol 3'-kinase inhibitor. GTPgammaS (guanosine 5'-3-O-(thio)triphosphate) increases the recycling of the chimera by 50% but has no effect on the recycling of the transferrin receptor. In these studies, we have identified in Chinese hamster ovary cells a novel, slow endocytic recycling mechanism that is regulated by insulin.
Johnson
A O
AO
Department of Biochemistry, Cornell University Medical College, New York, New York 10021, USA.
Subtil
A
A
Petrush
R
R
Kobylarz
K
K
Keller
S R
SR
McGraw
T E
TE
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D010122:Cystinyl Aminopeptidase; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers; D004705:Endocytosis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007328:Insulin / Q000502:physiology*; D007329:Insulin Antagonists / Q000494:pharmacology; D008969:Molecular Sequence Data; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011990:Receptors, Transferrin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; D017931:DNA Primers; D004791:Enzyme Inhibitors; D007328:Insulin; D007329:Insulin Antagonists; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000626:Aminopeptidases; D010122:Cystinyl Aminopeptidase; C105964:leucyl-cystinyl aminopeptidase; D000077191:Wortmannin