Transmembrane helix orientation influences membrane binding of the intracellular juxtamembrane domain in Neu receptor peptides

Abstract

The transmembrane (TM) and juxtamembrane (JM) regions of the ErbB family receptor tyrosine kinases connect the extracellular ligand-binding domain to the intracellular kinase domain. Evidence for the role of these regions in the mechanism of receptor dimerization and activation is provided by TM-JM peptides corresponding to the Neu (or rat ErbB2) receptor. Solid-state NMR and fluorescence spectroscopy show that there are tight interactions of the JM sequence with negatively charged lipids, including phosphatidylinositol 4,5-bisphosphate, in TM-JM peptides corresponding to the wildtype receptor sequence. We observe a release of the JM sequence from the negatively charged membrane surface using peptides containing an activating V664E mutation within the TM domain or in peptides engineered to form TM helix dimers with Val664 in the interface. These results provide the basis of a mechanism for coupling ligand binding to kinase activation in the full-length receptor.EGF receptor | HER2 receptor | PIP 2 T he receptor tyrosine kinases (RTKs) are a large family of membrane receptors that control cell growth, differentiation, and migration. These receptors have a three-domain architecture consisting of an extracellular ligand-binding domain, a single transmembrane helix, and an intracellular kinase domain. Receptor activation is triggered by ligand-mediated dimerization of receptor monomers or the structural rearrangement of inactive, preformed dimers (1, 2). Detailed information about the mechanisms of activation and regulation of RTKs has come largely from crystal structures of their extracellular and intracellular domains in inactive and active conformations. The activation mechanisms of the ErbB subfamily of RTKs are of particular interest because mutations and deletions that result in constitutive receptor activity have been identified in a number of human tumors (3, 4).In the epidermal growth factor receptor (EGFR), a member of the ErbB subfamily, the structures of the extracellular domain with (5) and without (6) bound EGF revealed that the unliganded structure has a tethered conformation that undergoes a dramatic rearrangement upon ligand binding. On the intracellular side of the EGFR, crystal structures show that the activation loop associated with the active site in the kinase domain is in an open, active conformation before ligand binding (7) or locked in a Src tyrosine kinase/cyclin-dependent kinase-like inactive conformation (8). Activation is produced not via phosphorylation of the activation loop, as in other RTKs, but through the association of the intracellular kinase domains as an asymmetric dimer (8). An open question has involved how structural changes induced by ligand binding lead to the formation of an active, asymmetric dimer on the intracellular side of the receptor.The single transmembrane (TM) helix and associated juxtamembrane (JM) sequences bridge the extracellular and intracellular domains and require a membrane environment to adopt their native structure. In general, ligand binding to the e...

The EGFR Family: Not So Prototypical Receptor Tyrosine Kinases

“…They showed that a peptide corresponding to the amino-terminal iJM region (residues 645-660) binds strongly to negatively charged membrane surfaces. Although they initially suggested that calmodulin binding to the iJM region might dissociate it from the membrane (and thus activate EGFR), more recent NMR studies have suggested that TM-domain dimerization or reorientation may be sufficient to dissociate the iJM region from the membrane, presumably by stabilizing the antiparallel a-helix dimer shown in Figure 3C (Matsushita et al 2013). These possibilities clearly need further investigation in the context of the intact receptor.…”

Thermodynamic and kinetic characterization of transmembrane helix association

“…[1][2][3] Interestingly, several helical membrane proteins require a stable or transient association for their function, and recent evidence points to a ligand-independent association kinetics. 4,5 High resolution microscopy methods have revealed new insights into monomer-dimer equilibrium within membranes, 6 but the link between the molecular interactions and the population behavior is still missing. Furthermore, conformational flexibility within the transmembrane dimer state has been shown to be functionally-relevant, 7,8 suggesting a complex nature of transmembrane helix association beyond the simple sequence motifs characterized earlier.…”

Basic Motifs Target PSGL-1, CD43, and CD44 to Plasma Membrane Sites Where HIV-1 Assembles

“…Basic sequences in the juxtamembrane domains of several transmembrane proteins are shown to interact with acidic phospholipids (67)(68)(69)(70)(71)(72)(73). Given that Gag can bind to acidic lipids, such as PI(4,5)P 2 and phosphatidylserine (PS), through the HBR (5, 12, 39), we speculated that clustering of acidic lipids at assembly sites by multimerizing Gag molecules may attract PSGL-1 and possibly also CD43 and CD44.…”