Jan S. Rosenbaum

Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells.

The bone morphogenetic proteins (BMPs) are a group of transforming growth factor beta (TGF-beta)-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-beta 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-beta and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP-4 binding and the appearance of a major affinity-labeled product of approximately 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. Images

Vascular Endothelial Growth Factor Receptor-2 and Neuropilin-1 Form a Receptor Complex That Is Responsible for the Differential Signaling Potency of VEGF165 and VEGF121

The two most abundant secreted isoforms of vascular endothelial growth factor A (VEGF165 and VEGF121) are formed as a result of differential splicing of the VEGF-A gene. VEGF165 and VEGF121 share similar affinities at the isolated VEGF receptor (VEGFR)-2 but have been previously demonstrated to have differential ability to activate VEGFR-2-mediated effects on endothelial cells. Herein we investigate whether the recently described VEGF165 isoform-specific receptor neuropilin-1 (Npn-1) is responsible for the difference in potency observed for these ligands. We demonstrate that although VEGFR-2 and Npn-1 form a complex, this complex does not result in an increase in VEGF165 binding affinity. Therefore, the differential activity of VEGF165 and VEGF121cannot be explained by a differential binding affinity for the complex. Using an antagonist that competes for VEGF165 binding at the VEGFR-2·Npn-1 complex, we observe specific antagonism of VEGF165-meditated phosphorylation of VEGFR-2 without affecting the VEGF121 response. These data indicate that the formation of the complex is responsible for the increased potency of VEGF165 versus VEGF121. Taken together, these data suggest a receptor-clustering role for Npn-1, as opposed to Npn-1 behaving as an affinity-converting subunit.

Identification of a Human Type II Receptor for Bone Morphogenetic Protein-4 That Forms Differential Heteromeric Complexes with Bone Morphogenetic Protein Type I Receptors

Bone morphogenetic proteins (BMPs) comprise the largest subfamily of TGF-β-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. When expressed alone in COS cells, BRK-3 specifically binds BMP-4, but cross-linking of BMP-4 to BRK-3 is undetectable in the absence of either the BRK-1 or BRK-2 BMP type I receptors. Cotransfection of BRK-2 with BRK-3 greatly enhanced affinity labeling of BMP-4 to the type I receptor, in contrast to the affinity labeling pattern observed with the BRK-1 + BRK-3 heteromeric complex. Furthermore, a subpopulation of super-high affinity binding sites is formed in COS cells upon cotransfection only of BRK-2 + BRK-3, suggesting that the different heteromeric BMP receptor complexes have different signaling potential.

AMD3100 Is a CXCR7 Ligand with Allosteric Agonist Properties

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 β-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.

Urothelial beta‐3 adrenergic receptors in the rat bladder

To investigate the distribution of beta‐3 adrenergic receptors (β3ARs) in the rat bladder and to examine the contribution of urothelial β3ARs to agonist‐induced suppression of bladder reflexes and relaxation of smooth muscle.

Effects of β3-Adrenergic Receptor Activation on Rat Urinary Bladder Hyperactivity Induced by Ovariectomy

Voiding dysfunctions, including increased voiding frequency, urgency, or incontinence, are prevalent in the postmenopausal population. β3-Adrenergic receptor (β3AR) 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 β3AR agonists to treat bladder hyperactivity in this setting. OVX increased voiding frequency and decreased bladder capacity by ∼25% in awake rats and induced irregular cystometrograms in urethane-anesthetized rats. Reverse transcription-polymerase chain reaction revealed three βARs subtypes (β1,2,3) in bladder tissue, and immunostaining indicated β3AR localization in urothelium and detrusor. Receptor expression was not different in OVX and SHAM rats. The β3AR agonist selectivity of BRL37344 [(±)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid sodium hydrate], TAK-677 [(3-((2R)-(((2R)-(3-chlorophenyl)-2-hydroxyethyl)amino)propyl)-1H-indol-7-yloxy)acetic acid], and FK175 [acetic acid, 2-[[(8S)-8-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]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 βARs. Intravenous injection of each of the β3AR agonists (0.1–500 μg/kg) in anesthetized rats decreased voiding frequency, bladder pressure, and amplitude of bladder contractions. In bladder strips, β3AR agonists (10-12-10-4 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 β3AR expression or the bladder inhibitory effects of β3AR agonists. This suggests that β3AR agonists should prove effective for the treatment of overactive bladder symptoms in the postmenopausal population.

Cannabinor, a Selective Cannabinoid-2 Receptor Agonist, Improves Bladder Emptying in Rats With Partial Urethral Obstruction

PURPOSE We studied the effects of chronic treatment with the novel selective cannabinoid 2 receptor agonist cannabinor (Procter & Gamble Pharmaceuticals, Cincinnati, Ohio) on bladder function in conscious rats with partial urethral obstruction and on the functional properties of isolated detrusor muscle. MATERIALS AND METHODS A total of 24 female Sprague-Dawley® rats with surgically created partial urethral obstruction received daily intraperitoneal injections of 3 mg/kg cannabinor (12) or saline as controls (12) for 2 weeks. Cystometry was done, the rats were sacrificed and the bladders were prepared for in vitro studies. RESULTS Mean ± SEM bladder weight was 0.97 ± 0.15 gm in controls and 0.53 ± 0.08 gm in cannabinor treated rats (p <0.05). There was no difference between the groups in the mean micturition interval, or mean baseline, threshold, flow or maximum pressure. In controls and cannabinor treated rats mean post-void residual volume was 0.28 ± 0.07 and 0.06 ± 0.02 ml, mean micturition compliance was 0.032 ± 0.006 and 0.069 ± 0.016 ml/cm H(2)O, and mean bladder wall force at the start of flow was 950 ± 280 and 1,647 ± 325 mN/gm, respectively (each p <0.05). Nonvoiding contractions were significantly less frequent in cannabinor treated rats than in controls. We noted no difference in carbachol (Sigma®) half maximum concentration between the groups but the carbachol maximum response in detrusor strips from cannabinor treated rats was significantly higher than that in control strips. CONCLUSIONS In rats with partial urethral obstruction treated daily for 14 days with cannabinor bladder weight was lower, the ability to empty the bladder was preserved and nonvoiding contraction frequency was low compared to those in controls. Detrusor preparations from cannabinor treated rats showed a higher response to nerve stimulation than those from controls. Selective cannabinoid 2 receptor activation may be a novel principle to enable improved bladder function after partial urethral obstruction.

Characterization of the RDC1 gene which encodes the canine omolog of a proposed human VIP receptor Expression does not correlate with an increase in VIP binding sites

We have isolated a portion of the canine gene encoding the orphan receptor RDC1 [1]. The complete coding sequence is contained in a single exon, and an intron divides the 5′ untranslated region of RDC1 mRNA. The RDC1 protein is 94% homologous to the gene product of GPRN1, which has been proposed to serve as a VIP receptor when expressed in CHO‐K1 and COS‐7 cells (Sreedharan, S.P. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 4986–4990). Northern analysis indicates that CHO‐K1 cells endogenously express a 2.1 kb RDC1 mRNA. However, while CHO‐K1 cells possess detectable low affinity [125I]VIP binding sites, VIP binding is not altered in membranes of CHO‐K1 cells expressing varying amounts of the RDC1 gene construct. Further, endogenous VIP binding is not increased by transient expression of RDC1 in COS‐7 cells. Taken together, the data suggest that RDC1 is not a canine homolog of the proposed VIP receptor.

The Y2 receptor mediates increases in collateral-dependent blood flow in a model of peripheral arterial insufficiency

We have utilized a rat model of peripheral artery disease (PAD) to examine whether the known angiogenic activity of the Y(2) receptor would translate into a meaningful increase in collateral blood flow. The maximal increase in collateral blood flow capacity of approximately 60% (p<0.001) was obtained with a 10microg/kgday (IA infusion, 14 days) of either PYY or PYY(3-36) and did not differ from that obtained with a maximally angiogenic dose of VEGF(165). Pharmacodynamic modeling based upon single dose pharmacokinetic plasma profiles of both agonists suggests that E(max) is reached when the Y(2) receptor is occupied by >or=50%. Furthermore, for PYY(3-36), occupancy of the Y(2) receptor is sufficient to promote a significant benefit in collateral blood flow.

CANNABINOR, A NOVEL PERIPHERALLY ACTING CANNABINOID-2-RECEPTOR AGONIST REDUCES NONVOIDING-CONTRACTIONS IN RATS WITH PARTIAL URETHRAL OBSTRUCTION

potentially contribute to bladder dysfunction. This study investigates if changes in caveolae or caveolar elements occur after bladder outlet obstruction (BOO), and whether these changes affect the responsiveness to agonists. METHODS: BOO was created for 1, 2 and 4 weeks by placing a ligature around the proximal urethra of male rats. Differences in bsm caveolae after BOO were visualized by electron microscopy. Changes in caveolin (Cav) expression and distribution after BOO were determined by real time rt-PCR and immunofluorescence microscopy. Longitudinal tissue from obstructed bladder was stretched in organ bath at 37oC. Contractile responses to physiologic agonists were repeated before and after disruption of caveolae (by methyl-s-cyclodextrin, mscd), and compared with non obstructed controls. RESULTS: After 2 weeks of BOO, bladder weight markedly increased compared to controls. The number of caveolae significantly decreased after 1 wk of BOO compared with non-obstructed bladders. Cav-1 immunoreactivity diminished after 1 wk of BOO and further decreased after 2 wks of BOO. Cav-2 immunoreactivity decreased after 1 wk of BOO but was less attenuated after 2-wks. Cav-2 gene expression was significantly up-regulated after 2wks of BOO. Following 2 wks of BOO, the contractile response to bradykinin (BK) did not change after caveolar depletion compared with non-obstructed animals, in which m cd significantly increased the BK-induced contraction; however in BOO animals the amplitude of BK contraction was greater at baseline compared with non obstructed controls. In non obstructed bladders, the contractile response to 5HT was diminished by caveolar depletion. In BOO, the 5HT induced response was slightly diminished by m cd after 2 weeks, but after 4 weeks the contractile response was not affected by m cd. CONCLUSIONS: This study linked the loss of caveolar elements in BOO with functional alteration of specific agonist-induced responses. These data are consistent with reduced negative and positive modulation of BK and 5HT induced contractions respectively, which normally is imparted by caveolae, but is progressively lost during BOO as caveolae gradually disappear from the bsm membrane. Thus, alterations in caveolae may play a role in bladder dysfunction induced by BOO.