The bicyclam AMD3100 is known as a small synthetic inhibitor of the CXCL12-binding chemokine receptor CXCR4. Here, we show that AMD3100 also binds to the alternative CXCL12 receptor CXCR7. CXCL12 or AMD3100 alone activate ␤-arrestin recruitment to CXCR7, which we identify as a previously unreported signaling pathway of CXCR7. In addition, AMD3100 increases CXCL12 binding to CXCR7 and CXCL12-induced conformational rearrangements in the receptor dimer as measured by bioluminescence resonance energy transfer. Moreover, small but reproducible increases in the potency of CXCL12-induced arrestin recruitment to CXCR7 by AMD3100 are observed. Taken together, our data suggest that AMD3100 is an allosteric agonist of CXCR7. The finding that AMD3100 not only binds CXCR4, but also to CXCR7, with opposite effects on the two receptors, calls for caution in the use of the compound as a tool to dissect CXCL12 effects on the respective receptors in vitro and in vivo.Chemokine receptors belong to the G-protein coupled receptor (GPCR) family. GPCR binding by ligands is believed to alter receptor conformation in a way that is transmitted to the cytoplasmic face of the receptor and triggers the activation of the heterotrimeric G-proteins as well as that of other G-protein-independent effectors. Although ligand-induced conformational change of the receptor has for long been deduced from functional data, the advent of new biophysical methods has eventually permitted direct measurement of such changes in native receptors present in the plasma membrane of live cells. bioluminescence resonance energy transfer (BRET) is one of the resonance energy transfer techniques used to show constitutive dimerization of chemokine receptors and other GPCRs (Terrillon and Bouvier, 2004). Constitutively dimeric GPCR BRET couples can also be used to probe receptor conformation, because BRET depends on the distance between the luminescence donor [Renilla reniformis luciferase (RLuc)] and the acceptor [the yellow fluorescent protein (YFP)]. For instance, the use of CXCR4-RLuc/ CXCR4-YFP dimers as sensors permitted the detection of different conformations in a panel of CXCR4 mutants (Berchiche et al., 2007). Moreover, dimeric BRET sensors permit the measurement of ligand-induced conformational changes in the receptor dimer (Ayoub et al., 2004;Percherancier et al., 2005). These effects are not only observed for cognate agonists but also for small synthetic ligands that may be orthosteric or allosteric modulators.Allosteric modulation of receptor-ligand interactions results from binding of a second (allosteric) ligand to a distinct site on the receptor, in a way that does not directly compete with binding of the cognate (orthosteric) ligand. Binding of the allosteric ligand may decrease the affinity of the cognate ligand, resulting in negative allosteric modulation. Conversely, the presence of the allosteric modulator may increase binding of the cognate ligand, called positive allosteric modulation (May et al., 2007). Allosteric ligands may also have ...

Neuropilin-1 (Npn-1) is a receptor for both semaphorin 3A (Sema3A) and vascular endothelial growth factor 165 (VEGF 165 ). To understand the role Npn-1 plays as a receptor for these structurally and functionally unrelated ligands, we set out to identify structural features of Npn-1 that confer binding to Sema3A or VEGF 165 . We constructed Npn-1 variants containing deletions within the "a" A complex but ordered series of axon guidance decisions during development is critical for the establishment of nervous system structure and function (1). The vertebrate vascular network is similarly complex, with interconnecting conduits that extend throughout the body that are often in close anatomical proximity to nerve pathways (2). Recent evidence suggests that at least some of the same ligand-receptor systems coordinate development of both the nervous system and the cardiovascular system. For example, Eph receptors and their ligands, the ephrins, were first characterized as mediators of repulsive guidance events crucial for correct navigation of neuronal growth cones and migrating neural crest cells (3, 4). Their unexpected role in blood vessel formation was revealed when mutant mice that lacked either ephrin B2 or its cognate receptor EphB4 were shown to die during embryogenesis due to cardiovascular dysfunction (5, 6). Consistent with this observation, ephrin B2 and EphB4 were shown to have a reciprocal expression patterns in arterial and venous endothelial cells (6).Another example of a cell surface receptor whose function is required for development of both the cardiovascular and nervous systems is neuropilin-1 (Npn-1).

The two most abundant secreted isoforms of vascular endothelial growth factor A (VEGF 165

The bone morphogenetic proteins (BMPs) are a group of transforming growth factor ,B (TGF-Pi)-related factors whose only receptor identified to date is the product of the daf-4 gene from Caenorhabditis elegans. Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57-to 62-kDa) and type II (75-to 82-kDa) receptor components for TGF-, and activin. Using a PCR approach, we have cloned a cDNA from NIH 3T3 cells which encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-,B and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP4 binding and the appearance of a major affinity-labeled product of -64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. We conclude that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs.The transforming growth factor ,B (TGF-,B) superfamily contains a large number of growth, differentiation, and morphogenetic cytokines that are active as homo-or heterodimers.This superfamily includes the TGF-1 family, the activin family, the Mullerian inhibiting substance (MIS), and the bone morphogenetic protein (BMP)/Vg family, which composes the largest group (reviewed in reference 39). Human BMP-2 and BMP-4 and their Drosophila homolog dpp are highly related (74 to 76% sequence identity [72]), as are BMP-5, BMP-6, and BMP-7 and their Drosophila homolog 60A (69 to 73% sequence identity [21]). Given the high degree of structural similarity among these family members, it is expected that their receptors will also form a family of related molecules. activin (2, 40) have been cloned. It has recently been determined that the product of the daf-4 gene from Caenorhabditis elegans, which is involved in both inhibition of dauer larva formation and exit from the dauer stage, is a type II receptor for BMP-2 and BMP-4 (24). Each of these receptors is predicted to be a transmembrane serine/threonine kinase. Ligand binding to each of these type II receptors in a variety of cell types does not appear to require coexpression of additional receptor components, indicating that type II receptors are capable of...

Bone morphogenetic proteins (BMPs) comprise the largest subfamily of TGF-␤-related ligands and are known to bind to type I and type II receptor serine/ threonine kinases. Although several mammalian BMP type I receptors have been identified, the mammalian BMP type II receptors have remained elusive. We have isolated a cDNA encoding a novel transmembrane serine/threonine kinase from human skin fibroblasts which we demonstrate here to be a type II receptor that binds BMP-4. This receptor (BRK-3) is distantly related to other known type II receptors and is distinguished from them by an extremely long carboxyl-terminal sequence following the intracellular kinase domain. The BRK-3 gene is widely expressed in a variety of adult tissues. Bone morphogenetic proteins (BMPs) 1 are the largest subfamily of growth factors in the TGF-␤ superfamily and have been demonstrated to play important roles in endochondral bone formation and embryogenesis (1-4). Like other members of the TGF-␤ superfamily, BMPs appear to interact with type I and type II receptors on the cell surface (5). Following the expression cloning of the type II receptors for activin and TGF-␤ (6, 7), an increasing number of transmembrane serine/ threonine (Ser/Thr) kinases have been identified in mammals based on the conserved amino acid sequences in the intracellular kinase domain (8, 9). These include three distinct mammalian type I receptors for BMPs that are distinguished from the type I receptors for TGF-␤ (10 -12) and activin (11, 13) by the capability of binding ligand on their own when transfected into COS cells (5,14,15). BRK-1 (also known as ALK-3, TFR11, and BMPR-IA) binds BMP-2 and BMP-4 more efficiently than it binds BMP-7 (5, 14, 15). BRK-2 (also known as ALK-6, BMPR-IB, and RPK-1 (16)) binds both BMP-4 and BMP-7 efficiently (15). ActRI (also known as ALK-2 and SKR1) binds both activin and BMP-7 but does not bind BMP-4 (15, 17).The only type II receptors that have been identified for BMPs to date are from non-mammalian sources. The product of the daf-4 gene from Caenorhabditis elegans binds both BMP-2 and BMP-4 (18) and forms a complex with each of the BMP type I receptors in the presence of ligand (5, 15). The product of the punt gene from Drosophila, originally identified as the Drosophila homologue of the activin type II receptor (AtrII (19)), has recently been demonstrated to bind BMP-2 and is required for signaling by the Drosophila homologue of BMP-2 and BMP-4, the product of the decapentaplegic gene (20,21). The ability of the mammalian BMP type I receptors to form a complex with the nematode Daf-4 type II receptor as well as with other proteins having the expected size of the type II receptor in endogenous systems (5, 15) suggests the existence of mammalian BMP type II receptors. Indeed, expression of a BMP type I receptor in COS cells is insufficient to reproduce the high affinity binding observed in endogenous systems, suggesting that the high affinity complex is composed of both type I and type II receptor subunits (5). Since complex f...