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The role of the antiapoptotic protein Bcl-xL in regulating mitochondrial Ca2+ ([Ca2+]mito) handling was examined in wild-type (WT) and Bcl-xL knock-out (Bcl-xL-KO) mouse embryonic fibroblast cells. Inositol 1,4,5-trisphosphate-generating agonist evoked cytosolic Ca2+ transients that produced a larger [Ca2+]mito uptake in WTcells compared with Bcl-xL-KO. In permeabilized cells, stepping external [Ca2+] from 0 to 3 μM also produced a larger [Ca2+]mito uptake in WT; moreover, the [Ca2+]mito uptake capacity of Bcl-xL-KO cells was restored by re-expression of mitochondrially targeted Bcl-xL. Bcl-xL enhancement of [Ca2+]mito uptake persisted after dissipation of the mitochondrial membrane potential but was absent in mitoplasts lacking an outer mitochondrial membrane. The outer membrane-localized voltage-dependent anion channel (VDAC) is a known Ca2+ permeability pathway that directly interacts with Bcl-xL. Bcl-xL interacted with VDAC1 and -3 isoforms, and peptides based on the VDAC sequence disrupted Bcl-xL binding. Peptides reduced [Ca2+]mito uptake inWTbut were without effect in Bcl-xL-KO cells. In addition, peptides reduced [Ca2+]mito uptake in VDAC1 and VDAC3 knock-out but not VDAC1 and -3 double knock-out mouse embryonic fibroblast cells, confirming that Bcl-xL interacts functionally with VDAC1 and -3 but not VDAC2. Thus, an interaction between Bcl-xL and VDAC promotes matrix Ca2+ accumulation by increasing Ca2+ transfer across the outer mitochondrial membrane. [ABSTRACT FROM AUTHOR]

We have provided the first evidence for specific heteromerization between the α1A-adrenoceptor (α1AAR) and CXC chemokine receptor 2 (CXCR2) in live cells. α1AAR and CXCR2 are both expressed in areas such as the stromal smooth muscle layer of the prostate. By utilizing the G protein-coupled receptor (GPCR) heteromer identification technology on the live cell-based bioluminescence resonance energy transfer (BRET) assay platform, our studies in human embryonic kidney 293 cells have identified norepinephrine-dependent β-arrestin recruitment that was in turn dependent upon co-expression of α1AAR with CXCR2. These findings have been supported by co-localization observed using confocal microscopy. This norepinephrine-dependent β-arrestin recruitment was inhibited not only by the α1AR antagonist Terazosin but also by the CXCR2-specific allosteric inverse agonist SB265610. Furthermore, Labetalol, which is marketed for hypertension as a nonselective β-adrenoceptor antagonist with α1AR antagonist properties, was identified as a heteromer-specific-biased agonist exhibiting partial agonism for inositol phosphate production but essentially full agonism for β-arrestin recruitment at the α1AAR-CXCR2 heteromer. Finally, bioluminescence resonance energy transfer studies with both receptors tagged suggest that α1AAR-CXCR2 heteromerization occurs constitutively and is not modulated by ligand. These findings support the concept of GPCR heteromer complexes exhibiting distinct pharmacology, thereby providing additional mechanisms through which GPCRs can potentially achieve their diverse biological functions. This has important implications for the use and future development of pharmaceuticals targeting these receptors. [ABSTRACT FROM AUTHOR]

Members of the Bcl-2 protein family play a central role in the regulation of apoptosis. An interaction between anti-apoptotic Bcl-xL and the endoplasmic reticulum (ER)-localized inositol trisphosphate receptor Ca2+ release channel (InsP3R) enables Bcl-xL to be fully efficacious as an anti-apoptotic mediator (White, C., Li, C., Yang, J., Petrenko, N. B., Madesh, M., Thompson, C. B., and Foskett, J. K. (2005) Nat. Cell Biol. 7, 1021-1028). Physiologically, Bcl-xL binds to the InsP3R to enhance its gating and Ca2+ signaling. Here we have discovered that structurally related proteins Bcl-2 and Mcl-1 function similarly. Bcl-2, Md1 and Bcl-xL bind with comparable affinity to the carboxyl termini of all three mammalian InsP3R isoforms with important functional consequences. Stable expression of Bcl-2 or Md-1 lowered ER Ca2+ content and enhanced the rate of InsP3-mediated Ca2+ release in response to submaximal InsP3 stimulation in permeabilized wild-type DT40 cells but not in cells lacking InsP3R. In addition, expression of either Bcl-2 or Mcl-1 enhanced spontaneous InsP3R-dependent Ca2+ oscillations and spiking in intact cells in the absence of agonist stimulation. Bcl-2 and Mcl-1-mediated protection from apoptosis induced by staurosporine or etoposide was enhanced in cells expressing InsP3R, demonstrating that their interactions with InsP3R enable Bcl-2 and Md-1 to be fully efficacious anti-apoptotic mediators, Our data suggest a molecular mechanism that is shared by several anti-apoptotic Bcl-2 proteins that provides apoptosis resistance by direct interactions at the ER with the InsP3R that impinges on cellular Ca2+ homeostasis. [ABSTRACT FROM AUTHOR]