Insulin and antibodies against insulin receptor cap on the membrane of cultured human lymphocytes

Abstract

When insulin interacts with its target cells, it binds to a specific membrane receptor and triggers a chain of events which stimulate many physiological responses^1,2. Although the mechanism of insulin action remains uncertain^2-4, electron microscopy has suggested that unoccupied insulin receptors appear in clusters of two to six molecules and that insulin becomes internalized after binding to them^5,6. Insulin receptors on 3T3 fibroblasts labelled with rhodamine–insulin (R-insulin) are diffusely distributed and mobile on the cell surface, with a lateral diffusion coefficient D∼(3–5)×10⁻¹⁰cm²s⁻¹. At 37°C, hormone–receptor complexes aggregate into immobile patches which are soon endocytosed^7,8. A new tool for such studies has been provided by the discovery of autoantibodies to the insulin receptor in the sera of certain patients with insulin-resistant diabetes⁹. These antibodies stimulate various physiological responses normally mediated by insulin^10-14. Both the bivalent antibodies (Fab′)₂ and the monovalent Fab′ block insulin binding, but only the bivalent antibodies are biologically active¹⁴, Fab′ acting as a competitive antagonist¹⁴. Similarly, antibodies raised against purified insulin receptors can mimic insulin¹⁵, suggesting that microaggregation of receptors could be involved in the action of this hormone^14,16. We now report that at 37°C both R-insulin and the rhodamine-labelled antibodies against insulin receptors form a single cap on one pole of the cell. Cells which were first treated at 37°C with fluorescein–insulin (F-insulin, at partial receptor occupancy), fixed with formaldehyde and then treated with either R–insulin or rhodamine-labelled anti-receptor antibodies, showed overlapping caps of the two markers. These experiments demonstrate directly the aggregation of insulin receptors and suggest either that insulin receptors are multivalent towards the hormone or that the unoccupied receptors migrate together with the occupied ones in the formation of caps.