Functional and structural differences in human and rat-derived insulin receptors: characterization of the beta-subunit kinase activity.
We studied the kinase activity of partially purified insulin receptor preparations from various rat and human tissues. Time courses for in vitro autophosphorylation were determined, and times to reach half-maximal (t1/2 max) and maximal (tmax) 32P incorporation were compared. Insulin receptors from rat muscle, liver, and fat had a t1/2 max of 7-10 min and a tmax of 60 min; human-derived insulin receptors had a t1/2 max in excess of 30 min and a tmax of 120 min. A spectrum of autophosphorylation time courses was present in human tissues; placenta-derived receptors exhibited a t1/2 max of 13 min while receptors from monocytes and fibroblasts had t1/2 max values of 60 and 80 min, respectively. The ATP Km for autophosphorylation of human-derived receptors was 5-fold greater than that of rat-derived receptors (266 +/- 27 vs. 48 +/- 8 microM, respectively). In contrast, when the receptors were first maximally prephosphorylated, the ATP Km values for substrate phosphorylation of human- and rat-derived receptors were equivalent (12.5 and 11.4 microM). Kact values for Mn were comparable in both human- and rat-derived adipocyte receptors. In addition to the functional differences between species, the apparent mol wt of the beta-subunit of rat-derived receptors (96,000) was consistently greater than that of human-derived receptor beta-subunits (93,000). In contrast to these in vitro findings, the ability of insulin to stimulate receptor kinase activity in isolated adipocytes was rapid, with a maximal effect by seconds. This was comparable for both rat and human tissues, suggesting that the in vitro autophosphorylation differences may not govern kinase activity in vivo.
Brillon
D J
DJ
Department of Medicine, University of California-San Diego, La Jolla 92093.
Henry
R R
RR
Klein
H H
HH
Olefsky
J M
JM
Freidenberg
G R
GR
D000273:Adipose Tissue / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D009132:Muscles / Q000378:metabolism; D009928:Organ Specificity; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism*; D013045:Species Specificity
D046911:Macromolecular Substances; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin