Bone morphogenetic proteins (BMPs) are multifunctional proteins regulating cell growth, differentiation, and apoptosis. BMP-2 signals via two types of receptors (BRI and BRII) that are expressed at the cell surface as homomeric as well as heteromeric complexes. Prior to ligand binding, a low but measurable level of BMP-receptors is found in preformed hetero-oligomeric complexes. The major fraction of the receptors is recruited into hetero-oligomeric complexes only after ligand addition. For this, BMP-2 binds first to the high affinity receptor BRI and then recruits BRII into the signaling complex. However, ligand binding to the preformed complex composed of BRII and BRI is still required for signaling, suggesting that it may mediate activating conformational changes. Using several approaches we have addressed the following questions: (i) Are preformed complexes incompetent of signaling in the absence of BMP-2? (ii) Which domains of the BRII receptors are essential for this complex formation? (iii) Are there differences in signals sent from BMP-induced versus preformed receptor complexes? By measuring the activation of Smads, of p38 MAPK and of alkaline phosphatase, we show that the ability of kinase-deficient BRII receptor mutants to inhibit BMP signaling depends on their ability to form heteromeric complexes with BRI. Importantly, a BRII mutant that is incapable in forming preassembled receptor complexes but recruits into a BMP-induced receptor complex does not interfere with the Smad pathway but does inhibit the induction of alkaline phosphatase as well as p38 phosphorylation. These results indicate that signals induced by binding of BMP-2 to preformed receptor complexes activate the Smad pathway, whereas BMP-2-induced recruitment of receptors activates a different, Smad-independent pathway resulting in the induction of alkaline phosphatase activity via p38 MAPK.Bone morphogenetic proteins (BMPs) 1 are members of the transforming growth factor-␤ (TGF-␤) superfamily that play important roles in most morphogenetic processes during development (1). BMPs are able to induce the formation of bone at nonbony sites in the adult animal by influencing the differentiation of mesenchymal progenitor cells along the cartilage lineage pathway. BMPs act on osteoblasts as well as chondrocytes but also on many other cell types such as neuronal cells (2, 3). Signaling by BMP-2 involves two types of transmembrane serine/threonine kinases, termed type I (BRI) and type II (BRII) receptors (4 -8). Receptors of both types are needed to form a functional complex to initiate further signaling events. Activated BMP type I receptors phosphorylate Smad1, Smad5, and Smad8 (R-Smads), which then assemble into heteromeric complexes with Smad4 (Co-Smad) and translocate into the nucleus to regulate transcription of target genes (9, 10). In addition, BMP receptors initiate other signaling pathways, distinct from the Smad pathway, resulting in the activation of p38 MAPK and JNK (11-13).We have shown recently that the oligomerization pattern of ...

Abstract. In this study we tested the hypothesis that fusion mediated by the influenza virus hemagglutinin (HA) is a cooperative event. To do this we characterized 3T3 cell lines that express HA at nine different defined surface densities. HA densities ranged from 1.0 to 12.6 × 103 HA trimers/~m 2 as determined by quantitative fluorescent antibody binding. The lateral mobility and percent mobile fraction of HA did not vary significantly among these cells, nor did the contact area between HA-expressing cells and target RBCs. The fusion reaction of each HA-expressing cell line was analyzed using a fluorescence dequenching assay that uses octadecylrhodamine (R18)-labeled RBCs. For each cell line we measured the lag time preceding the onset of fusion, the initial rate of fusion, and the final extent of fusion. The final extent of fusion was similar for all cell lines, and the initial rate of fusion as a function of HA surface density displayed a MichaelisMenten-type dependence. However, the dependence of the lag time preceding the onset of fusion on HA surface density was clearly sigmoidal. Kinetic analysis of the data for the reciprocal lag time vs HA surface density, by both a log/log plot and a Hill plot, suggested that the observed sigmoidicity does not reflect cooperativity at the level of formation of HA aggregates as a prerequisite to fusion. Rather, the cooperativity of the process(es) that occur(s) during the lag time arises at a later step and involves a minimum of three, and most likely four, HA trimers. A model is proposed to explain HA cooperativity during fusion.

Endocytosis is important for a variety of functions in eukaryotic cells, including the regulation of signaling cascades via transmembrane receptors. The internalization of bone morphogenetic protein (BMP) receptor type I (BRI) and type II (BRII) and its relation to signaling were largely unexplored. Here, we demonstrate that both receptor types undergo constitutive endocytosis via clathrin-coated pits (CCPs) but that only BRII undergoes also caveola-like internalization. Using several complementary approaches, we could show that (i) BMP-2-mediated Smad1/5 phosphorylation occurs at the plasma membrane in nonraft regions, (ii) continuation of Smad signaling resulting in a transcriptional response requires endocytosis via the clathrin-mediated route, and (iii) BMP signaling leading to alkaline phosphatase induction initiates from receptors that fractionate into cholesterol-enriched, detergent-resistant membranes. Furthermore, we show that BRII interacts with Eps15R, a constitutive component of CCPs, and with caveolin-1, the marker protein of caveolae. Taken together, the localization of BMP receptors in distinct membrane domains is prerequisite to their taking different endocytosis routes with specific impacts on Smad-dependent and Smad-independent signaling cascades.

H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopalmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cholesterol-dependent and cholesterol-independent microdomains. In contrast, monopalmitoylation of Cys181 dramatically reverses Hras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-palmitate is weaker. Thus, membrane affinity of a palmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopalmitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.Ras GTPases operate as plasma membrane-localized molecular switches that regulate multiple signal transduction pathways. The three ubiquitously expressed Ras isoforms, H-, N-, and K-ras, are anchored to the inner surface of the plasma membrane by a C-terminal S-farnesyl cysteine carboxy methylester acting in concert with a second signal. The S-farnesyl cysteine carboxy methylester is generated by a triplet of post-

The bone morphogenetic proteins (BMPs) play important roles in embryogenesis and normal cell growth. The BMP receptors belong to the family of serine/threonine kinase receptors, whose activation has been investigated intensively for the transforming growth factor-beta (TGF-beta) receptor subfamily. However, the interactions between the BMP receptors, the composition of the active receptor complex, and the role of the ligand in its formation have not yet been investigated and were usually assumed to follow the same pattern as the TGF-beta receptors. Here we demonstrate that the oligomerization pattern of the BMP receptors is different and is more flexible and susceptible to modulation by ligand. Using several complementary approaches, we investigated the formation of homomeric and heteromeric complexes between the two known BMP type I receptors (BR-Ia and BR-Ib) and the BMP type II receptor (BR-II). Coimmunoprecipitation studies detected the formation of heteromeric and homomeric complexes among all the BMP receptor types even in the absence of ligand. These complexes were also detected at the cell surface after BMP-2 binding and cross-linking. Using antibody-mediated immunofluorescence copatching of epitope-tagged receptors, we provide evidence in live cells for preexisting heteromeric (BR-II/BR-Ia and BR-II/BR-Ib) and homomeric (BR-II/BR-II, BR-Ia/ BR-Ia, BR-Ib/ BR-Ib, and also BR-Ia/ BR-Ib) oligomers in the absence of ligand. BMP-2 binding significantly increased hetero- and homo-oligomerization (except for the BR-II homo-oligomer, which binds ligand poorly in the absence of BR-I). In contrast to previous observations on TGF-beta receptors, which were found to be fully homodimeric in the absence of ligand, the BMP receptors show a much more flexible oligomerization pattern. This novel feature in the oligomerization mode of the BMP receptors allows higher variety and flexibility in their responses to various ligands as compared with the TGF-beta receptors.