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

β3-Adrenergic receptor agonists are currently under clinical development for the treatment of overactive bladder, a condition that is prevalent in postmenopausal women. These agents purportedly relax bladder smooth muscle through a direct action at the myocyte β3-receptor. The aim of this study was to examine the expression of the individual beta-adrenergic receptors in full thickness sections from ageing human female bladder. We obtained a series of rabbit polyclonal antibodies generated against each of the three β-adrenergic receptors, and validated their receptor specificity in CHOK1 cells expressing each of the individual receptors. Immunostaining for β1, β2, and β3 were each more prominent in the urothelium than in the detrusor, with all receptors expressed in the same cell types, indicating co-expression of all three receptors throughout the urothelium in addition to the detrusor. Staining of all receptors was also observed in suburothelial myofibroblast-like cells, intramural ganglion cells, and in Schwann cells of intramural nerves. The β3-receptor in the human urothelium appears to be functional, as two different selective β3-receptor agonists, TAK677 and BRL37344, stimulate cAMP formation in UROtsa cells. Densitometry analysis indicates a persistent expression of all receptors throughout the bladder with increasing age, with the exception of the β2-receptor in the urothelium of the trigone, which appears to decrease slightly in older women. These data indicate that β3-receptor expression is maintained with age, but may function in concert with other β-receptors. Activation of the myocyte receptor may be influenced by action on non-myocyte structures including the intramural ganglion cells and myofibroblasts.

Voiding dysfunctions, including increased voiding frequency, urgency, or incontinence, are prevalent in the postmenopausal population. ␤ 3 -Adrenergic receptor (␤ 3 AR) agonists, which relax bladder smooth muscle, are being developed to treat these conditions. We utilized the rat ovariectomy (OVX) model to investigate the effect of ovarian hormone depletion on bladder function and the potential for ␤ 3 AR agonists to treat bladder hyperactivity in this setting. OVX increased voiding frequency and decreased bladder capacity by ϳ25% in awake rats and induced irregular cystometrograms in urethane-anesthetized rats. Reverse transcription-polymerase chain reaction revealed three ␤ARs subtypes (␤ 1,2,3 ) in bladder tissue, and immunostaining indicated ␤ 3 AR localization in urothelium and detrusor. Receptor expression was not different in OVX and SHAM rats. ]amino]-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-yl]oxy], ethyl ester, hydrochloride] was confirmed by examining the relative potency for elevation of cAMP in CHOK1 cells overexpressing the various rat ␤ARs. Intravenous injection of each of the ␤ 3 AR agonists (0.1-500 g/kg) in anesthetized rats decreased voiding frequency, bladder pressure, and amplitude of bladder contractions. In bladder strips, ␤ 3 AR agonists (10 Ϫ12-10 Ϫ4 M) decreased baseline tone and reduced spontaneous contractions. BRL37344 (5 mg/kg) and TAK-677 (5 mg/kg) injected intraperitoneally in awake rats decreased voiding frequency by 40 to 70%. These effects were not altered by OVX. The results indicate that OVX-induced bladder dysfunction, including decreased bladder capacity and increased voiding frequency, is not associated with changes in ␤ 3 AR expression or the bladder inhibitory effects of ␤ 3 AR agonists. This suggests that ␤ 3 AR agonists should prove effective for the treatment of overactive bladder symptoms in the postmenopausal population.Lower urinary tract (LUT) dysfunctions, including increased voiding frequency, urgency, incontinence and nocturia, increase in the elderly population and following menopause (Stewart et al., 2003). These dysfunctions could result from hormonally induced changes in bladder contractile and/or relaxing mechanisms. Bladder contractions are triggered by parasympathetic nerves, which release ACh that in turn activates postjunctional muscarinic receptors (mAChRs) in the detrusor. Bladder relaxation is induced by release of norepinephrine from sympathetic nerves, which activates ␤-adrenergic receptors (␤AR) (Fowler et al., 2008