Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by bilateral schwannomas of the eighth cranial nerve. The NF2 tumor suppressor protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of membrane/F-actin linkers. Merlin resists solubilization by the detergent Triton X-100 (TX-100), a property commonly attributed to association with the cytoskeleton. Accordingly, NF2 patient mutations that encode merlins with enhanced TX-100 solubility have been explained previously in terms of loss of cytoskeletal attachment. However, here we present data to suggest that the detergent resistance of merlin is a result of its constitutive residence in lipid rafts. Furthermore, when cells are grown to high density, merlin shifts to a more buoyant lipid raft fraction in a density gradient. This shift is mimicked in subconfluent cells treated with cytochalasin D, suggesting that the shift results from merlin dissociation from the actin cytoskeleton, but not from lipid rafts. Intramolecular NH₂- and COOH-terminal binding, which occurs when merlin transitions to the growth-suppressive form, also brings about a similar change in buoyant density. Our results suggest that constitutive residence of merlin in lipid rafts is crucial for its function and that as merlin becomes growth suppressive in vivo, one significant molecular event may be the loss of interaction with the actin cytoskeleton. To our knowledge, merlin is the first tumor suppressor known to reside within lipid rafts, and the significance of this finding is underscored by known loss-of-function NF2 patient mutations that encode merlins with enhanced TX-100 solubility.

Farnesylation of Ras proteins is necessary for transforming activity. Although farnesyl transferase inhibitors show promise as anticancer agents, prenylation of the most commonly mutated Ras isoform, K-Ras4B, is difficult to prevent because K-Ras4B can be alternatively modified with geranylgeranyl (C20). Little is known of the mechanisms that produce incomplete or inappropriate prenylation. Among non-Ras proteins with CaaX motifs, murine guanylate-binding protein (mGBP1) was conspicuous for its unusually low incorporation of [³H]mevalonate. Possible problems in cellular isoprenoid metabolism or prenyl transferase activity were investigated, but none that caused this defect was identified, implying that the poor labeling actually represented incomplete prenylation of mGBP1 itself. Mutagenesis indicated that the last 18 residues of mGBP1 severely limited C20 incorporation but, surprisingly, were compatible with farnesyl modification. Features leading to the expression of mutant GBPs with partial isoprenoid modification were identified. The results demonstrate that it is possible to alter a protein's prenylation state in a living cell so that graded effects of isoprenoid on function can be studied. The C20-selective impairment in prenylation also identifies mGBP1 as an important model for the study of substrate/geranylgeranyl transferase I interactions.

Mutations inGJB1,the gene encoding the gap junction protein connexin32 (Cx32), cause the X-linked form of Charcot-Marie-Tooth disease, an inherited demyelinating neuropathy. The C terminus of human Cx32 contains a putative prenylation motif that is conserved in Cx32 orthologs. Using [³H]mevalonolactone ([³H]MVA) incorporation, we demonstrated that wild-type human connexin32 can be prenylated in COS7 cells, in contrast to disease-associated mutations that are predicted to disrupt the prenylation motif. We generated transgenic mice that express these mutants in myelinating Schwann cells. Male mice expressing a transgene were crossed with femaleGjb1-null mice; the male offspring were allGjb1-null, and one-half were transgene positive; in these mice, all Cx32 was derived from expression of the transgene. The mutant human protein was properly localized in myelinating Schwann cells in multiple transgenic lines and did not alter the localization of other components of paranodes and incisures. Finally, both the C280G and the S281x mutants appeared to “rescue” the phenotype ofGjb1-null mice, because transgene-positive male mice had significantly fewer abnormally myelinated axons than did their transgene-negative male littermates. These results indicate that Cx32 is prenylated, but that prenylation is not required for proper trafficking of Cx32 and perhaps not even for certain aspects of its function, in myelinating Schwann cells.