Agonist-induced formation of unproductive receptor-G12 complexes.

G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V2 receptors (V2R) associate with both Gs and G12 heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V2R-Gs complexes, V2R-G12 complexes are not destabilized by guanine nucleotides and do not promote G12 activation. Activating V2R does not lead to signaling responses downstream of G12 activation, but instead inhibits basal G12-mediated signaling, presumably by sequestering G12 heterotrimers. Overexpressing G12 inhibits G protein receptor kinase (GRK) and arrestin recruitment to V2R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G12 that are insensitive to nucleotides, suggesting that unproductive GPCR-G12 complexes are not unique to V2R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.

Michaelis-Menten Quantification of Ligand Signaling Bias Applied to the Promiscuous Vasopressin V2 Receptor.

Activation of G protein-coupled receptors by agonists may result in the activation of one or more G proteins and recruitment of arrestins. The extent of the activation of each of these pathways depends on the intrinsic efficacy of the ligand. Quantification of intrinsic efficacy relative to a reference compound is essential for the development of novel compounds. In the operational model, changes in efficacy can be compensated by changes in the "functional" affinity, resulting in poorly defined values. To separate the effects of ligand affinity from the intrinsic activity of the receptor, we developed a Michaelis-Menten based quantification of G protein activation bias that uses experimentally measured ligand affinities and provides a single measure of ligand efficacy. We used it to evaluate the signaling of a promiscuous model receptor, the Vasopressin V2 receptor (V2R). Using BRET-based biosensors, we show that the V2R engages many different G proteins across all G protein subfamilies in response to its primary endogenous agonist, arginine vasopressin, including Gs and members of the Gi/o and G12/13 families. These signaling pathways are also activated by the synthetic peptide desmopressin, oxytocin, and the nonmammalian hormone vasotocin. We compared bias quantification using the operational model with Michaelis-Menten based quantification; the latter accurately quantified ligand efficacies despite large difference in ligand affinities. Together, these results showed that the V2R is promiscuous in its ability to engage several G proteins and that its' signaling profile is biased by small structural changes in the ligand. SIGNIFICANCE STATEMENT: By modelling the G protein activation as Michaelis-Menten reaction, we developed a novel way of quantifying signalling bias. V2R activates, or at least engages, G proteins from all G protein subfamilies, including Gi2, Gz, Gq, G12, and G13. Their relative activation may explain its Gs-independent signalling.

180W-LBO GreenLight XPS laser vaporization for benign prostatic hyperplasia: our experience with current markers of surgical proficiency for durable and reproducible outcomes.

INTRODUCTION: This study aims at analyzing the impact of reaching current markers of proficiency on intra and postoperative clinical outcomes of laser vaporization with 180W GreenLight XPS in the treatment of benign prostatic hyperplasia. MATERIALS AND METHODS: A retrospective analysis was conducted on a prospectively collected database of 328 consecutive patients who underwent photoselective vaporization of the prostate (PVP) using Greenlight XPS performed by a single experienced laser surgeon. A logarithmic model was used to evaluate the case number to attain benchmark criteria for durable treatment. We compared clinical outcomes before and after current markers of proficiency, defined as either an energy density of 4kJ/cm³ or a 6 month prostate-specific antigen (PSA) drop of = 50%, were attained. RESULTS: Energy delivered per prostate volume increased significantly with experience. The published benchmark values of 4kJ/cm³ and 6 month PSA drop of 50% were attained after 190 and 155 cases, respectively. There were no significant differences between groups in intraoperative complications or postoperative functional outcomes. However, the number of Clavien-Dindo category I adverse events significantly decreased with experience. Sub-analysis evaluating prostate volumes ≤ 80 cm³ and > 80 cm³ demonstrated comparable clinical outcomes before and after technical proficiency. CONCLUSION: In our experience, the case volume required to achieve consistent reference values related to durable clinical outcomes and surgical proficiency was > 150 cases. However, desirable clinical outcomes were attained before reaching current markers of proficiency, regardless of preoperative prostate size. This suggests that current thresholds of technical proficiency may not be a good predictor of satisfying clinical outcomes.

Engagement of ß-arrestin by transactivated insulin-like growth factor receptor is needed for V2 vasopressin receptor-stimulated ERK1/2 activation.

G protein-coupled receptors (GPCRs) have been shown to activate the mitogen-activated protein kinases, ERK1/2, through both G protein-dependent and -independent mechanisms. Here, we describe a G protein-independent mechanism that unravels an unanticipated role for ß-arrestins. Stimulation of the V2 vasopressin receptor (V2R) in cultured cells or in vivo in rat kidney medullar collecting ducts led to the activation of ERK1/2 through the metalloproteinase-mediated shedding of a factor activating the insulin-like growth factor receptor (IGFR). This process was found to be both Src- and ß-arrestin-dependent. Whereas Src was found to act upstream of the metalloproteinase activation and be required for the release of the IGFR-activating factor, ß-arrestins were found to act downstream of the IGFR transactivation. Unexpectedly, the engagement of ß-arrestins by the IGFR but not by the V2R was needed to promote the vasopressin-stimulated ERK1/2 activation, indicating that a pool of ß-arrestins distinct from those ß-arrestins recruited to the V2R acts downstream of the receptor tyrosine kinase to activate ERK1/2. Such a dual site of action for ß-arrestins helps explain the pleiotropic actions of this scaffolding protein. Given the role that V2R-stimulated ERK1/2 plays in kidney cell proliferation, this transactivation mechanism may have important implications for renal pathophysiology. Still, the role of ß-arrestins downstream of a transactivation event is not limited to the V2R, because we observed a similar involvement for an unrelated GPCR (the platelet-activating factor receptor), indicating that it may be a general mechanism shared among GPCRs.

Multiplexing of multicolor bioluminescence resonance energy transfer.

Bioluminescence resonance energy transfer (BRET) is increasingly being used to monitor protein-protein interactions and cellular events in cells. However, the ability to monitor multiple events simultaneously is limited by the spectral properties of the existing BRET partners. Taking advantage of newly developed Renilla luciferases and blue-shifted fluorescent proteins (FPs), we explored the possibility of creating novel BRET configurations using a single luciferase substrate and distinct FPs. Three new (to our knowledge) BRET assays leading to distinct color bioluminescence emission were generated and validated. The spectral properties of two of the FPs used (enhanced blue (EB) FP2 and mAmetrine) and the selection of appropriate detection filters permitted the concomitant detection of two independent BRET signals, without cross-interference, in the same cells after addition of a unique substrate for Renilla luciferase-II, coelentrazine-400a. Using individual BRET-based biosensors to monitor the interaction between G-protein-coupled receptors and G-protein subunits or activation of different G-proteins along with the production of a second messenger, we established the proof of principle that two new BRET configurations can be multiplexed to simultaneously monitor two dependent or independent cellular events. The development of this new multiplexed BRET configuration opens the way for concomitant monitoring of various independent biological processes in living cells.