MANNOSE 6-PHOSPHATE INSULIN-LIKE GROWTH-FACTOR-II RECEPTOR FAILS TO INTERACT WITH G-PROTEINS - ANALYSIS OF MUTANT CYTOPLASMIC-RECEPTOR DOMAINS

The binding of insulin-like growth factor II (IGF II) to the mannose 6-phosphate (M6P)/IGF II receptor has previously been reported to induce the activation of trimeric G(12) proteins by functional coupling to a 14-amino acid region within the cytoplasmic receptor domain (Nishimoto, I., Murayama, Y,, Hatada, T,, Ui, M,, and Ogata, E, (1989) J, Biol, Chem, 264, 14029-14038), In the present study, we examined further the potential functional coupling of G-proteins with the human M6P/IGF II receptor and mutant receptors lacking the proposed G-protein activator sequence, IGF II treatment of mouse L-cells expressing either wild type or mutant M6P/IGF II receptors failed to attenuate the pertussis toxin-catalyzed modification of a 40-kDa protein or enhance GTPase activity, In broken L-cell membranes expressing wild type or mutant M6P/IGF II receptors, 30 nM IGF II also failed to affect the pertussis toxin substrate activity, By using phospholipid vesicles reconstituted with human wild type or mutant M6P/IGF II receptors and pertussis toxin-sensitive G-proteins, no stimulation of GTP gamma S binding to or GTPase activity of G(i2), G(o1), or G(i)/G(o) mixtures were observed in response to 1 mu M IGF II, Furthermore, in vesicles containing purified wild type M6P/IGF II receptors and monomeric G alpha(o1) or G alpha(i2), and beta gamma dimers no effects of IGF II on GTP gamma S binding could be detected, However, when vesicles reconstituted with M6P/IGF II receptors and G(i2) proteins were incubated with 100 mu M mastoparan GTP gamma S binding was stimulated and GTPase activity was increased significantly, These results indicate that the human M6P/IGF II receptor neither interacts with G proteins in mouse L-cell membranes nor is coupled to G(i2) proteins in phospholipid vesicles, This study suggests strongly that the M6P/IGF II receptor does not function in transmembrane signaling in response to IGF II.