The juxtamembrane but not the carboxyl-terminal domain of the insulin receptor mediates insulin's metabolic functions in primary adipocytes and cultured hepatoma cells

Insulin-stimulated signaling pathways are activated upon interactions betwe en the intracellular domains of the receptor and its downstream effecters. Insulin receptor substrate proteins (IRS-1, -2, -3 and -4) are the best-stu died substrates for the insulin receptor kinase (IRK). We have previously s hown that IRS-1 and IRS-2 interact with the juxtamembrane (JM) but not with the carboxyl-terminal (CT) region of the insulin receptor (IR) in vitro. H owever, the precise role of these IR regions in mediating insulin's bioeffe cts is still unresolved. In the present work we made use of vaccinia virus as a vector for quantitative expression of the JM and CT domains within the cytoplasm of physiologically insulin-responsive primary rat adipocytes and rat hepatoma Fao cells. We could demonstrate that overexpression of either the JM or the CT domains did not inhibit either insulin binding or insulin -stimulated receptor autophosphorylation. In contrast, metabolic effects su ch as insulin-induced glucose utilization in adipocytes, and insulin-induce d amino acid utilization in Fao hepatoma cells were inhibited (70-80%) in c ells over-expressing the JM but not the CT domains of IR. The inhibitory ef fects of the overexpressed JM domain were accompanied by inhibition of insu lin-stimulated IRS-1 phosphorylation, decreased IRS-1-associated PI3K activ ity, and decreased phosphorylation of the downstream effectors of PI3K, PKB and p70 S6K. Insulin-stimulated thymidine incorporation in Fao cells was a lso inhibited (40%) upon overexpression of the JM but not the CT region of IR. Our findings suggest that interactions between the JM region of IR and its downstream effecters are obligatory for insulin-stimulated metabolic fu nctions in physiologically relevant insulin responsive cells. They also rul e out the possibility that interaction of proteins, including PI3K, with th e CT domain can provide an alternative pathway.