Insulin-induced circular membrane ruffling on rat 1 cells expressing a high number of human insulin receptors: circular ruffles caused by rapid actin reorganization exhibit high density of insulin receptors and phosphotyrosines.
The early effects of insulin on morphological changes were examined using Rat1 cells expressing approximately one million human insulin receptors (Rat1HIR cells). Insulin transiently induced circular membrane ruffling on Rat1HIR cells. The circular ruffles were detected by phase microscopy as well as by fluorescence microscopy when actin was stained with rhodamine-conjugated phalloidin. The circular ruffles were also visualized by fluorescence microscopy when stained with either anti-insulin receptor monoclonal antibody, alpha IR-1, or anti-phosphotyrosine antibody, followed by fluorescein isothiocyanate-labeled second antibodies. Control Rat1 cells or Rat1HIR A/K1018 cells expressing kinase-defective insulin receptors did not show any circular ruffles when treated with insulin. These results suggest that the circular ruffles are formed by actin reorganization induced by insulin and that this process requires the protein-tyrosine kinase activity of the receptor. Detection of insulin receptors and phosphotyrosines in the circular ruffles suggests the possibility that the insulin receptors in the ruffles may be highly active and phosphorylating either the receptors themselves or proteins associated with the ruffles. These results are consistent with our previous in vitro observations that a large aggregate form of the purified insulin receptor is a highly active protein-tyrosine kinase.
Li
S L
SL
Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Miyata
Y
Y
Yahara
I
I
Fujita-Yamaguchi
Y
Y
D008841:Actin Cytoskeleton / Q000502:physiology; D000199:Actins / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology*; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D011940:Receptor Aggregation; D011972:Receptor, Insulin / Q000502:physiology*; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D000199:Actins; D007328:Insulin; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin