Changes in insulin-receptor structure associated with trypsin-induced activation of the receptor tyrosine kinase

Abstract

The tyrosine kinase of the insulin receptor can be activated by trypsin treatment. The concomitant abolition of insulin binding has been postulated to result from proteolytic destruction of the receptor. A discrepancy between the decrease in insulin binding and receptor immunoreactivity after trypsin treatment led us to investigate more closely the structure of the trypsin-treated receptor. After trypsin treatment of the CHOT cell line, which over-expresses transfected human insulin receptors, insulin binding was significantly decreased, but reactivity with five alpha-subunit monoclonal antibodies was either unaffected or only moderately decreased, indicating that the alpha-subunit was substantially intact. Examination of receptor structure after trypsin treatment, receptor autophosphorylation and gel electrophoresis revealed a single band at 110 kDa in non-reduced gels, comprising a small fragment (21 kDa) of the alpha-subunit linked to the beta-subunit by class II disulphides. When the receptor was radio-labelled with 125I, two additional alpha-subunit bands of 142 kDa and 81 kDa (composed of identical reduced bands) were observed on non-reduced gels, which contained disulphide-linked (class I) fragments. All fragments could be precipitated by antibodies to both alpha- and beta-subunits. However, only antibodies directed towards the N-terminus of the receptor could immunoblot trypsin-treated fragments. Thus activation of the receptor tyrosine kinase by trypsin occurs after cleavage, but not loss of the alpha-subunit. This finding has implications for the mechanism of transmembrane activation of the receptor kinase by insulin.

Anti-apoptotic signaling of the IGF-I receptor in fibroblasts following loss of matrix adhesion

“…This discrepancy may be largely due to di erent experimental procedures: in the previous report, cells were trypsinized and then kept in suspension for 30 min. In our experiments, cells were detached by Versene, since, as mentioned above, trypsinization is known to partially digest the extracellular portions of growth factor receptors (Clark et al, 1991). Also at variance with other experimental situations, we found a lack of correlation with the serine/threonine kinase Akt, activation which has also been shown to be important in protection from apoptosis, including IGF-I mediated protection (Kennedy et al, 1997;Khwaja et al, 1997;Datta et al, 1997).…”

“…In preliminary experiments (not shown), we had observed that detachment of cells by trypsinization (as it is usually done also in our laboratory) caused the results to be somewhat erratic, probably due to the fact that trypsinization, even modest, induces partial breakdown of the extracellular part of growth factor receptors (Clark et al, 1991). p6 cells (mouse embryo ®broblasts that over-express the human IGF-IR) were chosen for the initial experiments, because they are known to form colonies in soft agar very e ciently (Pietrzkowski et al, 1992), and to grow in anchorageindependent conditions in vivo .…”

Brefeldin A inhibits insulin‐dependent receptor redistribution in HIRcB cells

“…As reported by other investigators, only receptors exposed to the extracellular medium are accessible to trypsin [4,23 26]. This treatment proteolyzes the ~-subunit of the receptor in a unique site close to the C-terminus of the ~-subunit and separates both subunits [27]. The larger fragment resulting from this treatment (100 kDa) contains both the mAb 83-7 and insulin binding sites, whereas the smaller fragment remains attached to the fl-subunit of the receptor [27].…”

“…These antibodies have been extensively described by Clark et al [27]. Anti-phosphotyrosine antibody 4GI0 was purchased from Upstate Biotechnologies.…”

“…Internalized receptors were distinguished from those present on the cell surface on the basis of their sensitivity to mild trypsin treatment of the cells, as originally described by Levy and Olefsky [24], a procedure that has been used as a tool to distinguish cell surface from internalized receptors [4,[23][24][25][26]. Mild trypsin treatment (1 mg/ml trypsin for 30 min at 4°C) cleaves the ~-subunit of the receptor predominantly at one site, releasing two fragments: a 100 kDa fragment that contains the binding sites for insulin and for the monoclonal antibody 83-7, and a 20 kDa fragment that remains attached to the fl-subunit [27]. Thus, the criterion used to detect intact receptors was the co-immunoprecipitation of c~-and fl-subunits: only those fl-subunits attached to intact, non-proteolyzed c~-subunits coimmunoprecipitate with mAb 83-7.…”