Superactive mutants of thromboxane prostanoid receptor: functional and computational analysis of an active form alternative to constitutively active mutants

Abstract

In class A GPCRs the E/DRY motif is critical for receptor activation and function. According to experimental and computational data, R3.50 forms a double salt bridge with the adjacent E/D3.49 and E/D6.30 in helix 6, constraining the receptor in an inactive state. The disruption of this network of interactions facilitates conformational transitions that generate a signal or constitutive activity. Here we demonstrate that non-conservative substitution of either E129((3.49)) or E240((6.30)) of thromboxane prostanoid receptor (TP) resulted in mutants characterized by agonist-induced more efficient signaling properties, regardless of the G protein coupling. Results of computational modeling suggested a more effective interaction between G(q) and the agonist-bound forms of the TP mutants, compared to the wild type. Yet, none of the mutants examined revealed any increase in basal activity, precluding their classification as constitutively active mutants. Here, we propose that these alternative active conformations might be identified as superactive mutants or SAM.

Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

“…4 (workflow). Briefly, structures of the single domains were obtained by homology modeling and assembled through an integrative modeling procedure consisting of hierarchical, rigid-body docking calculations carried out by means of the ZDOCK algorithm (60) using established protocols (61)(62)(63). Structural models obtained from global docking approaches were scored and filtered by applying cross-link-derived distance filters, using 35-Å and 31-Å thresholds for DSS and DSG, respectively (64).…”

Interruption of the Ionic Lock in the Bradykinin B2 Receptor Results in Constitutive Internalization and Turns Several Antagonists into Strong Agonists

“…The data also demonstrate that a high affinity state of a GPCR does not necessarily imply constitutive activity. Similarly, E6.30 mutations in the thromboxane prostanoid receptor resulted in more efficient agonist-induced signaling without any increase in basal activity (Ambrosio et al, 2010). Moreover, for the B 2 R, even the small molecule compound JSM10292, which thus far had displayed no partial agonistic activity (Faussner et al, 2012), becomes a full agonist in mutant E6.30R.…”

Light on the structure of thromboxane A2 receptor heterodimers

“…Receptor expression was monitored by equilibrium mixed type binding curves of [ 3 H]SQ 29,548 (48 Ci/mmol) performed as previously described [37,38]. Briefly, confluent adherent cells in 250 ll of serum-free DMEM, containing 0.2% (w/v) BSA were assayed in the presence of 0.1-1 nM serial dilutions of [ 3 H]SQ 29,548 and 3 nM-10 lM of the homologous cold ligand.…”

“…Total inositol phosphate production Signaling of TPs was assessed by measuring the accumulation of total IPs as previously described [37,38]. Briefly, cells labeled overnight with 0.5 lCi of myo-[2-3 H]inositol (18 Ci/mmol) were pre-incubated with 25 mM LiCl for 10 min followed by incubation with increasing concentrations of the TP selective U46619 agonist for 30 min at 37°C.…”