Regulatory Mechanisms Involved In Insulin-Induced Receptor Capping And Cell Activation In Lymphoid Cells

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Cell Biology and Anatomy


A variety of cyto- and biochemical techniques were used to examine the regulation of insulin-induced receptor capping in lymphoid cells. Immunofluorescence microscopy revealed that insulin induced cap structures morphologically similar to those induced by multivalent ligands and that these surface cap structures corresponded to accumulations of other membrane proteins, as well as intracellular actomyosin and contractile regulatory molecules such as calmodulin and myosin light chain kinase. Myosin was detected in an insulin receptor-cytoskeleton complex and could be co-immunoprecipitated with insulin receptors, further indicating the association of these molecules. In addition, insulin stimulated the phosphorylation of myosin light chains, indicating the functional role of insulin in activating the actomyosin contractile process responsible for the aggregation of insulin receptors.The functional relevance of insulin receptor capping was explored in mitogenically activated splenic T lymphocytes. Insulin receptor capping was correlated both temporally and morphologically with insulin-stimulated increase of glucose uptake and co-accumulation of receptors with glucose transport proteins. Since this process did appear to be physiologically relevant, we examined early insulin-stimulated signal transductory events that could play a role in the regulation of receptor capping. Prior to capping or glucose uptake, insulin induced an increase of intracellular calcium concentration, which was preceded by an increase of the mediator molecule IP$\sb3$. Preliminary data suggested that insulin regulated the appearance of IP$\sb3$ by affecting the level of precursor lipid PIP$\sb2$, as well as by affecting the activity of the cleavage enzyme phospholipase C via a GTP binding protein as has been suggested for other hormone-receptor systems. Therefore, insulin-induced receptor capping appeared to be regulated by an insulin-stimulated increase of calcium, via IP$\sb3$, which proceeded to activate calcium-calmodulin-regulated systems, such as myosin light chain kinase, responsible for activating the actomyosin contactile system involved in directing receptor movement, and this capping process may play a significant role in subsequent insulin-stimulated metabolic events.


Chemistry, Biochemistry

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