Petra Knaus

The Mode of Bone Morphogenetic Protein (BMP) Receptor Oligomerization Determines Different BMP-2 Signaling Pathways

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-inducedversus 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.

Recent advances in BMP receptor signaling

Bone Morphogenetic Proteins (BMPs) play an important role during organ development and during regeneration after tissue damage. BMPs signal via transmembrane serine/threonine kinase receptors. From our current understanding heteromeric complexes of type I and type II receptors are required for signal propagation. Presently, three type I and three type II receptors are known to bind BMPs with different affinities. Ligands and receptors eventually oligomerize via defined modes into signaling complexes. Co-receptors recruit into these complexes to either inhibit or to promote signaling. The Smad pathway, initiated by phosphorylation through the activated type I receptors, results in transcriptional regulation of early target genes. However, on its way to the nucleus, Smads represent signaling platforms for other pathways, which eventually finetune BMP signal transduction. We also describe BMP-induced signaling cascades leading to cytoskeletal rearrangements, non-transcriptional and non-Smad pathways. BMPs induce a plethora of different cellular effects ranging from stem cell maintenance, migration, differentiation, proliferation to apoptosis. The molecular mechanism, by which the same ligand induces these manifold effects, depends on the cellular context. Here we try to give a current picture of the most important players in regulating and directing BMP signaling towards the desired cellular outcome. Examples of BMP action during development, but also physiological and pathophysiological conditions in the adult organism are presented.

Different Routes of Bone Morphogenic Protein (BMP) Receptor Endocytosis Influence BMP Signaling

ABSTRACT 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.

Synaptophysin: molecular organization and mRNA expression as determined from cloned cDNA.

Synaptophysin is a major glycoprotein of Mr approximately 38,000 (in deglycosylated form: Mr approximately 34,000) characteristic of a certain class of small (30‐80 nm diameter) neurosecretory vesicles, including presynaptic vesicles, but also vesicles of various neuroendocrine cells of both neuronal and epithelial phenotype. Using synaptophysin‐specific antibodies we have isolated cDNA clones from rat nervous tissue libraries, which identify an approximately 2.5‐kb mRNA in rat and human cells, including neuroendocrine tumours, that contains a reading frame for a polypeptide of 307 amino acids with a total mol. wt of 33 312. The deduced amino acid sequence, which was partly confirmed by comparison with sequences of two tryptic peptides obtained from purified synaptophysin, revealed four hydrophobic regions of 24 amino acids each, which are characterized, according to conformation prediction analyses, by marked alpha‐helicity. The sequence shows a single potential N‐glycosylation site, which is assigned to the vesicle interior, and a carboxy‐terminal tail of 89 amino acids which contains glycine‐rich tetrapeptide repeats, the epitope of monoclonal antibody SY38, and a number of collagenase‐sensitive sites accessible on the surface of the intact vesicles. These features suggest that the polypeptide spans the vesicle membrane four times, with both N and C termini located on the outer, i.e. cytoplasmic, surface of the vesicles.

Integration of the TGF-β pathway into the cellular signalling network

Abstract Transforming growth factor-βs (TGF-βs) regulate pivotal cellular processes such as proliferation, differentiation and apoptosis. After ligand binding, the signals are transmitted by two types of transmembrane serine/threonine kinase receptors. The type I receptor phosphorylates Smad proteins, intracellular effectors which upon oligomerization enter the nucleus to regulate transcription following assembly with transcriptional co-factors and co-modulators. The cellular distribution of TGF-β receptors along with their oligomerization mode and their complex formation with different cell surface receptors represent crucial steps in determining the initiation of distinct signalling cascades. In addition, the broad array of intracellular proteins that influence the TGF-β pathway demonstrates that signal transduction does not proceed in a linear fashion but rather comprises a complex network of cascades that mutually influence each other. The present review describes the intricate control of TGF-β signal transduction on various levels of the cascade with particular focus (i) on the assembly of different receptor subtypes and (ii) on the multitude of crosstalk with signal transducers from other pathways. Integration of the TGF-β/Smad pathway into the signalling network has taken on added importance as it substantially contributes to elicit the plethora of cell- and tissue-specific effects of TGF-β.

Mutations in bone morphogenetic protein receptor 1B cause brachydactyly type A2

Brachydactyly (BD) type A2 is an autosomal dominant hand malformation characterized by shortening and lateral deviation of the index fingers and, to a variable degree, shortening and deviation of the first and second toes. We performed linkage analysis in two unrelated German families and mapped a locus for BD type A2 to 4q21-q25. This interval includes the gene bone morphogenetic protein receptor 1B (BMPR1B), a type I transmembrane serinethreonine kinase. In one family, we identified a T599 → A mutation changing an isoleucine into a lysine residue (I200K) within the glycine/serine (GS) domain of BMPR1B, a region involved in phosphorylation of the receptor. In the other family we identified a C1456 → T mutation leading to an arginine-to-tryptophan amino acid change (R486W) in a highly conserved region C-terminal of the BMPR1B kinase domain. An in vitro kinase assay showed that the I200K mutation is kinase-deficient, whereas the R486W mutation has normal kinase activity, indicating a different pathogenic mechanism. Functional analyses with a micromass culture system revealed a strong inhibition of chondrogenesis by both mutant receptors. Overexpression of mutant chBmpR1b in vivo in chick embryos by using a retroviral system resulted either in a BD phenotype with shortening and/or missing phalanges similar to the human phenotype or in severe hypoplasia of the entire limb. These findings imply that both mutations identified in human BMPR1B affect cartilage formation in a dominant-negative manner.

BMPs: from bone to body morphogenetic proteins.

Discussion at a meeting in Berlin, Germany, showed that BMPs have essential functions in organs and tissues besides bone. The family of bone morphogenetic proteins (BMPs) comprises approximately 30 secreted cytokines that signal through transmembrane serine/threonine kinase receptors. The BMP signaling pathways are fine-tuned on multiple levels: Extracellular antagonists modify ligand activity; several co-receptors enhance or inhibit downstream signaling events through multiple mechanisms; and intracellular molecules further regulate the signaling outcome and mediate crosstalk with other pathways. BMPs affect structures and processes throughout the entire body, ranging from embryonic patterning and development through stem cells and their niches, to tissue homeostasis and regeneration. This comprehensive involvement in various tissues had not been expected by Marshall Urist, who initially discovered the ability of an unknown factor in bone to induce bone growth in muscle and subsequently suggested the name “bone morphogenetic protein.” Today, recombinant BMPs are used in clinical practice for the treatment of bone and kidney disorders, and new genetically modified BMPs are emerging as promising tools in regenerative medicine and tissue engineering. Clearly, the functions of BMPs within the body are more versatile than initially suspected. To discuss modern trends in BMP signaling, leaders in the field met for the First International BMP Workshop in Berlin in September 2009. Here, we summarize new insights on the roles of BMPs in various tissues and highlight recent findings in cell, structural, and developmental biology as well as the therapeutic potential of BMPs. Finally, we conclude that BMPs today deserve to be called body morphogenetic proteins.

Dysregulated Bone Morphogenetic Protein Signaling in Monocrotaline-Induced Pulmonary Arterial Hypertension

Background—Mutations in the bmpr2 gene, encoding the type II bone morphogenetic protein (BMP) receptor, have been identified in patients with pulmonary arterial hypertension (PAH), implicating BMP signaling in PAH. The aim of this study was to assess BMP signaling and its physiological effects in a monocrotaline (MCT) model of PAH. Methods and Results—Expression of BMP receptors Ib and II, and Smads 4, 5, 6, and 8, was downregulated in lungs but not kidneys of MCT-treated rats. Smad1 phosphorylation and expression of BMP/Smad target genes id1 and id3 was also reduced, although ERK1/2 and p38MAPK phosphorylation remained unaffected. BMP receptor and Smad expression, Smad1 phosphorylation, and induction of the BMP/Smad-responsive element of the id1 promoter were reduced in pulmonary artery smooth muscle cells (PASMCs) from MCT-treated rats. As a consequence of impaired BMP/Smad signaling, PASMCs from MCT-treated rats were resistant to apoptosis induced by BMP-4 and BMP-7, and were also resistant to BMP-4 antagonism of proliferation induced by platelet-derived growth factor. Conclusion—BMP signaling and BMP-regulated physiological phenomena are perturbed in MCT-treated rats, lending solid support to the proposed roles for BMP signaling in the pathogenesis of human PAH.

Synaptoporin, a Novel Putative Channel Protein of Synaptic Vesicles

By homology screening of a rat brain library, we have isolated cDNAs that encode a novel member of the synaptophysin/connexin channel protein superfamily. The deduced protein, named synaptoporin, displays 58% amino acid identity to synaptophysin, with highly conserved transmembrane segments, but a divergent cytoplasmic tail. Northern blot analysis and PCR amplification of RNA from different rat tissues indicate expression of synaptoporin transcripts in the CNS. Antibodies against a synthetic peptide or a fusion construct encompassing the cytoplasmic tail region of synaptoporin detect a polypeptide of 37 kd that copurifies with small synaptic vesicles. Our data suggest the existence of a family of vesicular channel proteins whose members may be differently distributed among synaptic vesicle subpopulations.

A Particle-Associated Glycoprotein Signal Peptide Essential for Virus Maturation and Infectivity

ABSTRACT Signal peptides (SP) are key determinants for targeting glycoproteins to the secretory pathway. Here we describe the involvement in particle maturation as an additional function of a viral glycoprotein SP. The SP of foamy virus (FV) envelope glycoprotein is predicted to be unusually long. Using an SP-specific antiserum, we demonstrate that its proteolytic removal occurs posttranslationally by a cellular protease and that the major N-terminal cleavage product, gp18, is found in purified viral particles. Analysis of mutants in proposed signal peptidase cleavage positions and N-glycosylation sites revealed an SP about 148 amino acids (aa) in length. FV particle release from infected cells requires the presence of cognate envelope protein and cleavage of its SP sequence. An N-terminal 15-aa SP domain with two conserved tryptophan residues was found to be essential for the egress of FV particles. While the SP N terminus was found to mediate the specificity of FV Env to interact with FV capsids, it was dispensable for Env targeting to the secretory pathway and FV envelope-mediated infectivity of murine leukemia virus pseudotypes.