A highly phosphorylated subpopulation of insulin-like growth factor II/mannose 6-phosphate receptors is concentrated in a clathrin-enriched plasma membrane fraction.

Insulin-like growth factor II (IGF-II)/mannose 6-phosphate (Man-6-P) receptors immunoprecipitated from purified plasma membranes of 32P-labeled rat adipocytes are markedly heterogenous in their phosphorylation state. Approximately 80% of the plasma membrane receptors are solubilized in 1% (vol/vol) Triton X-100 and are phosphorylated on serine residues at a stoichiometry of approximately 0.1-0.2 mol of phosphate per mol of receptor. In contrast, 15-20% of the receptors are Triton X-100-insoluble and are phosphorylated on serine and threonine residues at approximately 4 or 5 mol of phosphate per mol of receptor. This Triton X-100-insoluble membrane subfraction contains only 5% of the total plasma membrane protein and yet contains all of the clathrin heavy chain associated with plasma membrane, as detected by immunoblotting with a monoclonal antibody. Based on the relative yields of protein in the detergent-insoluble material, IGF-II/Man-6-P receptors are concentrated approximately equal to 3-fold in this clathrin-enriched subfraction. Insulin treatment of intact cells increased the total IGF-II/Man-6-P receptors in the Triton X-100-soluble fraction of the plasma membrane, whereas no change in receptor number in the detergent-insoluble fraction was seen. However, insulin markedly decreased the phosphorylation stoichiometry of the Triton X-100-insoluble receptors. Taken together, these results indicate that insulin decreases the phosphorylation state of a highly phosphorylated subpopulation of IGF-II/Man-6-P receptors on the plasma membrane. In addition, insulin action may prevent the concentration of these receptors in a clathrin-enriched membrane subfraction

The role of the I(sK) protein in the specific pharmacological properties of the I(Ks) channel complex

I(Ks) channels are composed of I(sK) and KvLQT1 subunits and underly the slowly activating, voltage-dependent I(Ks) conductance in heart. Although it appears clear that the I(sK) protein affects both the biophysical properties and regulation of I(Ks) channels, its role in channel pharmacology is unclear. In the present study we demonstrate that KvLQT1 homopolymeric K(+) channels are inhibited by the I(Ks) blockers 293B, azimilide and 17-β-oestradiol. However, I(Ks) channels induced by the coexpression of I(sK) and KvLQT1 subunits have a 6–100 fold higher affinity for these blockers. Moreover, the I(Ks) activators mefenamic acid and DIDS had little effect on KvLQT1 homopolymeric channels, although they dramatically enhanced steady-state currents through heteropolymeric I(Ks) channels by arresting them in an open state. In summary, the I(sK) protein modulates the effects of both blockers and activators of I(Ks) channels. This finding is important for the action and specificity of these drugs as I(sK) protein expression in heart and other tissues is regulated during development and by hormones