Despite the importance of lipid rafts, commonly defined as liquid-ordered domains rich in cholesterol and in lipids with high gel-to-fluid melting temperatures (T(m)), the rules for raft formation in membranes are not completely understood. Here, a fluorescence-quenching strategy was used to define how lipids with low T(m), which tend to form disordered fluid domains at physiological temperatures, can stabilize ordered domain formation by cholesterol and high-T(m) lipids (either sphingomyelin or dipalmitoylphosphatidylcholine). In bilayers containing mixtures of low-T(m) phosphatidylcholines, cholesterol, and high-T(m) lipid, the thermal stability of ordered domains decreased with the acyl-chain structure of low-T(m) lipids in the following order: diarachadonyl > diphytanoyl > 1-palmitoyl 2-docosahexenoyl = 1,2 dioleoyl = dimyristoleoyl = 1-palmitoyl, 2-oleoyl (PO). This shows that low-T(m) lipids with two acyl chains having very poor tight-packing propensities can stabilize ordered domain formation by high-T(m) lipids and cholesterol. The effect of headgroup structure was also studied. We found that even in the absence of high-T(m) lipids, mixtures of cholesterol with PO phosphatidylethanolamine (POPE) and PO phosphatidylserine (POPS) or with brain PE and brain PS showed a (borderline) tendency to form ordered domains. Because these lipids are abundant in the inner (cytofacial) leaflet of mammalian membranes, this raises the possibility that PE and PS could participate in inner-leaflet raft formation or stabilization. In bilayers containing ternary mixtures of PO lipids, cholesterol, and high-T(m) lipids, the thermal stability of ordered domains decreased with the polar headgroup structure of PO lipids in the order PE > PS > phosphatidylcholine (PC). Analogous experiments using diphytanoyl acyl chain lipids in place of PO acyl chain lipids showed that the stabilization of ordered lipid domains by acyl chain and headgroup structure was not additive. This implies that it is likely that there are two largely mutually exclusive mechanisms by which low-T(m) lipids can stabilize ordered domain formation by high-T(m) lipids and cholesterol: 1), by having structures resulting in immiscibility of low-T(m) and high-T(m) lipids, and 2), by having structures allowing them to pack tightly within ordered domains to a significant degree.

Genetic disorders of cholesterol biosynthesis result in accumulation of cholesterol precursors and cause severe disease. We examined whether cholesterol precursors alter the stability and properties of ordered lipid domains (rafts). Tempo quenching of a raft-binding fluorophore was used to measure raft stability in vesicles containing sterol, dioleoylphosphatidylcholine, and one of the following ordered domain-forming lipids/lipid mixtures: dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), a SM/cerebroside mixture or a SM/ceramide (cer) mixture. Relative to cholesterol, early cholesterol precursors containing an 8 -9 double bond (lanosterol, dihydrolanosterol, zymosterol, and zymostenol) only weakly stabilized raft formation by SM or DPPC. Desmosterol, a late precursor containing the same 5-6 double bond as cholesterol, but with an additional 24 -25 double bond, also stabilized domain formation weakly. In contrast, two late precursors containing 7-8 double bonds (lathosterol and 7-dehydrocholesterol) were better raft stabilizers than cholesterol. For vesicles containing SM/cerebroside and SM/cer mixtures the effect of precursor upon raft stability was small, although the relative effects of different precursors were the same. Using both detergent resistance and a novel assay involving fluorescence quenching induced by certain sterols we found cholesterol precursors were displaced from cer-rich rafts, and could displace cer from rafts. Precursor displacement by cer was inversely correlated to precursor raft-stabilizing abilities, whereas precursor displacement of cer was greatest for the most highly raft-stabilizing precursors. These observations support the hypothesis that sterols and cer compete for raft-association

A variety of tumors contain activating mutations in the cytoplasmic juxtamembrane domain of the type III family of receptor-tyrosine kinases, and some constructed mutations in this domain induce ligand-independent receptor activation. To explore the role of this domain in regulation of receptor activity, we subjected the juxtamembrane domain of the murine platelet-derived growth factor (PDGF) ␤ receptor to alanine-scanning mutagenesis. The mutant receptors were expressed in Ba/F3 cells and tested for constitutive tyrosine phosphorylation, association with phosphatidylinositol 3-kinase, and their ability to induce cell survival and proliferation in the absence of interleukin-3. The mutant receptors accumulated to similar levels and appeared to undergo a normal PDGF-induced increase in tyrosine phosphorylation. Alanine substitutions at numerous positions located throughout the juxtamembrane domain caused constitutive receptor activation, as did an alanine insertion in the membrane-proximal segment of the juxtamembrane domain and a six-amino acid deletion in the center of the domain. It is possible to model the PDGF receptor juxtamembrane domain as a short ␣-helix followed by a three-stranded ␤-sheet very similar to the known structures of WW domains. Strikingly, the activating mutations clustered in the central portions of the first and second ␤ strands and along one face of the ␤-sheet, whereas the loops connecting the strands were largely devoid of mutationally sensitive positions. These findings provide strong support for the model that the activating mutations in the juxtamembrane region stimulate receptor activity by disrupting an inhibitory WWlike domain. Receptor tyrosine kinases (RTKs)1 are transmembrane proteins that regulate numerous aspects of cell physiology including proliferation and survival. Binding of a soluble ligand to the extracellular domain of these receptors typically induces receptor dimerization and trans-phosphorylation of the cytoplasmic catalytic domain. This tyrosine phosphorylation stimulates the intrinsic tyrosine kinase activity of the receptor and generates binding sites for signaling proteins containing SH2 domains. Although ligand-induced dimerization is an important trigger of receptor activation, receptor activity is also subject to additional levels of regulation. For example, the cytoplasmic juxtamembrane region of receptor tyrosine kinases, which is located between the transmembrane domain and the kinase domain, has been implicated in regulation of receptor enzymatic activity (e.g. see Ref.

The conjugated phenyltetraene PTE-ET-18-OMe (all-(E)-1-O-(15'-phenylpentadeca-8',10',12',14'-tetraenyl)-2-O-methyl-rac-glycero-3-phosphocholine) is a recently developed fluorescent lysophospholipid analog of edelfosine, (Quesada et al. (2004) J. Med. Chem. 47, 5333-5335). We investigated the use of this analog as a probe of membrane structure. PTE-ET-18-OMe was found to have several properties that are favorable for fluorescence anisotropy (polarization) experiments in membranes, including low fluorescence in water and moderately strong association with lipid bilayers. PTE-ET-18-OMe has absorbance and fluorescence properties similar to those of diphenylhexatriene (DPH) probes, with about as large a difference between its fluorescence anisotropy in liquid disordered (Ld) and ordered states (gel and Lo) as observed for DPH. Also like DPH, PTE-ET-18-OMe has a moderate affinity for both gel state ordered domains and Lo state ordered domains (rafts). However, unlike fluorescent sterols or DPH (Megha and London (2004) J. Biol. Chem. 279, 9997-10004), PTE-ET-18-OMe is not displaced from ordered domains by ceramide. Also unlike DPH, PTE-ET-18-OMe shows only slow exchange between the inner and outer leaflets of membrane bilayers, and can thus be used to examine anisotropy of an individual leaflet of a lipid bilayer. Since PTE-ET-18-OMe is a zwitterionic molecule, it should not be as influenced by electrostatic interactions as are other probes that do not cross the lipid bilayer but have a net charge. We conclude that PTE-ET-18-OMe has some unique properties that should make it a useful fluorescence probe of membrane structure.