Evidence supporting a passive role for the insulin receptor transmembrane domain in insulin-dependent signal transduction.
We previously have demonstrated that intramolecular interactions between alpha beta-alpha beta subunits are necessary for insulin-dependent activation of the protein kinase domain within a single alpha 2 beta 2 heterotetrameric insulin-receptor complex (Wilden, P. A., Morrison, B. D., and Pessin, J. E. (1989) Biochemistry 28, 785-792). To evaluate the role of the beta subunit transmembrane domain in the insulin-dependent signalling mechanism, mutant human insulin receptors containing a series of nested transmembrane domain deletions (amino acids 941-945) were generated and stable Chinese hamster ovary-transfected cell lines were obtained. In addition, a substitution of Val-938 for Glu (E/V938) similar to the oncogenic mutation found in the neu transmembrane domain was also introduced into the insulin receptor. Scatchard analysis of insulin binding to the stable Chinese hamster ovary cell lines expressing either wild type or mutant insulin receptors indicated equivalent receptor number (2-4 x 10(6)/cell) and similar high affinity binding constants (Kd 0.1-0.3 nM). 125I-Insulin affinity cross-linking demonstrated that all of the expressed insulin receptors were assembled and processed into alpha 2 beta 2 heterotetrameric complexes. Surprisingly, all the mutant insulin receptors retained insulin-stimulated autophosphorylation both in vivo and in vitro. Furthermore, endogenous substrate phosphorylation in vivo as well as insulin-stimulated thymidine incorporation into DNA were unaffected by the transmembrane domain mutations. These data demonstrate that marked structural alterations in the insulin receptor transmembrane domain do not interfere with insulin-dependent signal transduction.
Frattali
A L
AL
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242.
Treadway
J L
JL
Pessin
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D011972:Receptor, Insulin / Q000502:physiology*; D015398:Signal Transduction; D013379:Substrate Specificity; D013936:Thymidine / Q000378:metabolism
D003432:Cross-Linking Reagents; D007328:Insulin; D012333:RNA, Messenger; D011972:Receptor, Insulin; D013936:Thymidine