SYNTHESIS AND CHARACTERIZATION OF N-PARINAROYL ANALOGS OF GANGLIOSIDE-G(M3) AND DE-N-ACETYL G(M3) - INTERACTIONS WITH THE EGF RECEPTOR KINASE

A specific plasma membrane glycosphingolipid, known as ganglioside G(M 3), can regulate the intrinsic tyrosyl kinase activity of the epiderma l growth factor (EGF) receptor; this modulation is not associated with alterations in hormone binding to the receptor. G(M3) inhibits EGF re ceptor tyrosyl kinase activity in detergent micelles, in plasma membra ne vesicles, and in whole cells. In addition, immunoaffinity-purified EGF receptor preparations contain ganglioside G(M3) (Hanai et al. (198 8) J. Biol. Chem. 263, 10915-10921), implying that the glycosphingolip id is intimately associated with the receptor kinase in cell membranes . Both the nature of this association and the molecular mechanism of k inase inhibition remain to be elucidated. In this report, we describe the synthesis of a fluorescent analog of ganglioside G(M3), in which t he native fatty acid was replaced with trans-parinaric acid. This glyc osphingolipid inhibited the receptor kinase activity in a manner simil ar to that of the native ganglioside. A modified fluorescent glycosphi ngolipid, N-trans-parinaroyl de-N-acetyl ganglioside G(M3), was also p repared. This analog, like the nonfluorescent de-N-acetyl ganglioside G(M3), had no effect on receptor kinase activity. Results from tryptop han fluorescence quenching and steady-state anisotropy measurements in membranes containing these fluorescent probes and the human EGF recep tor were consistent with the notion that G(M3), but not de-N-acetyl G( M3), interacts specifically with the receptor in intact membranes.