Distinct alpha-subunit structures of human insulin receptor A and B variants determine differences in tyrosine kinase activities.
Human insulin receptor isoforms (HIR-A and -B) differ in their alpha-subunit structures which result from alternatively spliced precursor mRNAs. This structural difference causes distinct binding affinities for insulin. To determine the impact of the structural difference on receptor signaling, we characterized the tyrosine kinase activity of HIR-A and HIR-B in vitro and determined the insulin stimulated beta-subunit phosphorylation and tyrosine kinase activation in the intact cell. When 32P incorporation in beta-subunits of equal amounts of isolated HIR-A and HIR-B was measured, an increased 32P incorporation in tyrosine residues of the beta-subunit of HIR-B (2.5-fold) compared to that of HIR-A was found after in vitro insulin stimulation. This was paralleled by an increased rate of phosphorylation (2.0-fold) or poly(GluNa,Tyr 4:1). In vitro analysis of Km values for ATP were similar for HIR-A (Km = 14.3 microM +/- 3.8) and HIR-B (Km = 20.2 microM +/- 8.6), whereas the Vmax of HIR-B was significantly increased (HIR-A Vmax = 5.5 mumol/60 min micrograms-1 +/- 1.4, HIR-B Vmax = 42.5 mumol/60 min micrograms-1 +/- 19.2). HPLC analysis of tryptic beta-subunit phosphopeptides revealed identical patterns, suggesting that the difference in kinase activities is not due to an alteration of the phosphorylation-activation cascade within the beta-subunit. However, when cleavage of the alpha-subunit by short-time trypsinization was used to activate the tyrosine kinase, the differences in 32P incorporation between HIR-A and HIR-B were abolished.(ABSTRACT TRUNCATED AT 250 WORDS)
Kellerer
M
M
Institut für Diabetesforschung, München, Germany.
Lammers
R
R
Ermel
B
B
Tippmer
S
S
Vogt
B
B
Obermaier-Kusser
B
B
Ullrich
A
A
Häring
H U
HU
D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000737:chemistry / Q000201:enzymology; D014644:Genetic Variation; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine; D011233:Precipitin Tests; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D014357:Trypsin / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry
D007328:Insulin; D010446:Peptide Fragments; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D014357:Trypsin