One of the assumptions of the mobile receptor hypothesis as it relates to G protein-coupled receptors is that the stoichiometry of receptor, G protein, and effector is 1:1:1 (Bourne, H. R., Sanders, D. A., and McCormick, F. (1990) Nature 348, 125-132). Many studies on the cooperativity of agonist binding are incompatible with this notion and have suggested that both G proteins and their associated receptors can be oligomeric. However, a clear physical demonstration that G protein-coupled receptors can indeed interact as dimers and that such interactions may have functional consequences was lacking. Here, using differential epitope tagging we demonstrate that  2 -adrenergic receptors do form SDSresistant homodimers and that transmembrane domain VI of the receptor may represent part of an interface for receptor dimerization. The functional importance of dimerization is supported by the observation that a peptide derived from this domain that inhibits dimerization also inhibits -adrenergic agonist-promoted stimulation of adenylyl cyclase activity. Moreover, agonist stimulation was found to stabilize the dimeric state of the receptor, while inverse agonists favored the monomeric species, which suggests that interconversion between monomeric and dimeric forms may be important for biological activity.
The cytoskeleton-membrane linker protein ezrin has been shown to associate with phosphatidyl-inositol 4,5-bisphosphate (PIP2)-containing liposomes via its NH2-terminal domain. Using internal deletions and COOH-terminal truncations, determinants of PIP2 binding were located to amino acids 12–115 and 233–310. Both regions contain a KK(X)nK/RK motif conserved in the ezrin/radixin/moesin family. K/N mutations of residues 253 and 254 or 262 and 263 did not affect cosedimentation of ezrin 1-333 with PIP2-containing liposomes, but their combination almost completely abolished the capacity for interaction. Similarly, double mutation of Lys 63, 64 to Asn only partially reduced lipid interaction, but combined with the double mutation K253N, K254N, the interaction of PIP2 with ezrin 1-333 was strongly inhibited. Similar data were obtained with full-length ezrin. When residues 253, 254, 262, and 263 were mutated in full-length ezrin, the in vitro interaction with the cytoplasmic tail of CD44 was not impaired but was no longer PIP2 dependent. This construct was also expressed in COS1 and A431 cells. Unlike wild-type ezrin, it was not any more localized to dorsal actin-rich structures, but redistributed to the cytoplasm without strongly affecting the actin-rich structures. We have thus identified determinants of the PIP2 binding site in ezrin whose mutagenesis correlates with an altered cellular localization.
Actin assembly on membrane surfaces is an elusive process in which several phosphoinositides (PIPs) have been implicated. We have reconstituted actin assembly using a defined membrane surface, the latex bead phagosome (LBP), and shown that the PI(4,5)P 2 -binding proteins ezrin and/or moesin were essential for this process (Defacque et al., 2000b). Here, we provide several lines of evidence that both preexisting and newly synthesized PI(4,5)P 2 , and probably PI(4)P, are essential for phagosomal actin assembly; only these PIPs were routinely synthesized from ATP during in vitro actin assembly. Treatment of LBP with phospholipase C or with adenosine, an inhibitor of type II PI 4-kinase, as well as preincubation with anti-PI(4)P or anti-PI(4,5)P 2 antibodies all inhibited this process. Incorporation of extra PI(4)P or PI(4,5)P 2 into the LBP membrane led to a fivefold increase in the number of phagosomes that assemble actin. An ezrin mutant mutated in the PI(4,5)P 2 -binding sites was less efficient in binding to LBPs and in reconstituting actin assembly than wild-type ezrin. Our data show that PI 4-and PI 5-kinase, and under some conditions also PI 3-kinase, activities are present on LBPs and can be activated by ATP, even in the absence of GTP or cytosolic components. However, PI 3-kinase activity is not required for actin assembly, because the process was not affected by PI 3-kinase inhibitors. We suggest that the ezrin-dependent actin assembly on the LBP membrane may require active turnover of D4 and D5 PIPs on the organelle membrane. INTRODUCTIONA significant fraction of the de novo nucleation of actin in cells occurs on the cytoplasmic surface of eukaryotic cell membranes, especially the plasma membrane (Tilney, 1976;Carraway and Carraway, 1989;Small et al., 1995;Mitchison and Cramer, 1996), and a role for phosphoinositides in this elusive process has been widely discussed (Divecha and Irvine, 1995;Martin, 1998;Caroni, 2001). However, the precise function of these lipids is still not clear and is likely to be quite complicated. In several cellular systems that show rapid actin assembly in response to extracellular ligands, synthesis of phosphoinositides, especially phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ], and in some cases phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P 3 ], coincides precisely with the transient burst of actin assembly (Eberle et al., 1990;Dobos et al., 1992;Apgar, 1995;Hartwig et al., 1995;Gachet et al., 1997). In addition, overexpression of phosphatidylinositol-4-phosphate [PI(4)P] 5-kinase in cells leads to a significant polymerization of actin (Shibasaki et al., 1997). However, in other systems, the synthesis of PI(4,5)P 2 as well as PI(3,4,5)P 3 coincides more with actin depolymerization, after a transient assembly of F-actin (Apgar, 1995;Gratacap et al., 1998).One important clue to the functions of phosphoinositides in actin assembly/disassembly is that these lipids can bind in vitro to an increasing number of actin-binding proteins (ABPs). Interestingly, two different...
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