Characterization of insulin receptor substrate 4 in human embryonic kidney 293 cells.
We recently cloned IRS-4, a new member of the insulin receptor substrate (IRS) family. In this study we have characterized IRS-4 in human embryonic kidney 293 cells, where it was originally discovered. IRS-4 was the predominant insulin-elicited phosphotyrosine protein in these cells. Subcellular fractionation revealed that about 50% of IRS-4 was located in cellular membranes, and immunofluorescence indicated that IRS-4 was concentrated at the plasma membrane. Immunoelectron microscopy conclusively established that a large portion of the IRS-4 was located at the cytoplasmic surface of the plasma membrane in both the unstimulated and insulin-treated states. IRS-4 was found to be associated with two src homology 2 (SH2) domain-containing proteins, phosphatidylinositol 3-kinase and Grb2, the adaptor to the guanine nucleotide exchange factor for Ras. On the other hand, no significant association was detected with two other SH2 domain proteins, the SH2-containing protein tyrosine phosphatase 2 and phospholipase Cgamma. Insulin-like growth factor I acting through its receptor was as effective as insulin in eliciting tyrosine phosphorylation of IRS-4, but interleukin 4 and epidermal growth factor were ineffective.
Fantin
V R
VR
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Sparling
J D
JD
Slot
J W
JW
Keller
S R
SR
Lienhard
G E
GE
Lavan
B E
BE
D048868:Adaptor Proteins, Signal Transducing; D002460:Cell Line; D006133:Growth Substances / Q000378:metabolism; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D006133:Growth Substances; C107686:IRS4 protein, human; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D014443:Tyrosine