Results: 5

1.
Fig. 3

Fig. 3. From: Kinetic Scaffolding Mediated by a Phospholipase C-? and Gq Signaling Complex.

Secondary Gαq•PLC-β3 interface. (A) Ribbon diagram highlighting residues (gray) preceding the C2 domain (light blue) of PLC-β3 that interact with Sw1 and 2 (pink) of activated Gαq (green). AlF4 (gray cross-stick), Mg2+ (orange ball), and the catalytic water (red ball) within the nucleotide-binding pocket also are shown. (B) Mutational analysis of the Gαq•PLC-β3 binding interface. (Top) Activation of the indicated mutants of PLC-β3 in the presence of cotransfected Gαq (red) or Gβ1γ2 (gray) was determined by quantification of [3H]inositol phosphate accumulation in COS-7 cells. Data are mean ± SEM from four independent experiments. (Bottom) Relative expression of PLC-β3 and mutant forms was quantified under each transfection condition by using a PLC-β3–specific antibody. Actin immunoblots (IB) included as loading controls.

Gary L. Waldo, et al. Science. ;330(6006):974-980.
2.
Fig. 4

Fig. 4. From: Kinetic Scaffolding Mediated by a Phospholipase C-? and Gq Signaling Complex.

Interaction of the EF3/4 loop of PLC-β3 with switch residues critical for GTP hydrolysis by Gαq. (A) Ribbon and cylinder diagram highlighting conserved interactions within EF hands 3 and 4 (yellow) of PLC-β3 needed for the optimal positioning of Asn260 (red arrow) within the guanine nucleotide binding pocket of Gαq. Sw1 to Sw3 are pink; other portions of Gαq are green. The C2 domain and adjacent Hα1/Hα2 of PLC-β3 are light blue; key PLC-β3 residues (sticks) and hydrogen bonds (dotted lines) that support Asn260 (red arrow) are highlighted. The guanine nucleotide binding pocket contains GDP and AlF4 (sticks) as well as the Mg2+ cofactor (orange ball) and catalytic water (red ball). (B) Sequence alignment comparing EF hands 3 and 4 of PLC-βs with equivalent region of PLC-δ1. α helices (cylinders) and β sheets (arrows) assigned from crystal structures (PLC-β3, 3OHM; PLC-β2, 2ZKM; δ1, 1DJX); dashed lines bracket disordered regions. The asparagine (Asn260 in PLC-β3) that is positioned for promotion of GTP hydrolysis by Gαq is indicated by a red arrow. The colors correspond to those of the structures depicted in (D) below. Dots indicate every 10th residue. (C) Comparison of the GTP-binding sites of Gαq•PLC-β3 and Gαt•RGS9. Left image depicts portions of the EF3/4 loop (yellow) of PLC-β3 contacting Sw1 and 2 (light red) of Gαq. Other portions of Gαq are shown as in (A). Middle image highlights electron density (composite simulated annealing omit map contoured at 1.2 σ) centered on Asn260 of Gαq•PLC-β3. Right image depicts analogous portions of RGS9 (yellow) bound to Gαt as revealed in the crystal structure determined by Slep et al. (23). (D) Ribbon and cylinder diagrams comparing EF hands 3 and 4 of PLC-β3 (yellow) with PLC-β2 (top, magenta) and PLC-δ1 (bottom, purple). Asn260 highlighted with red arrow, and dotted lines indicate disordered portions of PLC-β2.

Gary L. Waldo, et al. Science. ;330(6006):974-980.
3.
Fig. 5

Fig. 5. From: Kinetic Scaffolding Mediated by a Phospholipase C-? and Gq Signaling Complex.

Contribution of the EF hand region to GAP activity of PLC-β3. (A) The GAP activity of purified wild-type PLC-β3 is compared with that of mutant PLC-β3 isozymes. Steady-state GTP hydrolysis was quantified with phospholipid vesicles reconstituted with purified P2Y1 receptor, Gαq, and Gβ1γ2. Assays were in the presence of the P2Y1 receptor agonist 2MeSADP (3 μM) and the indicated concentrations of purified PLC-β3; PLC-β3(δEF); PLC-β3(V262A); or PLC-β3(N260A), PLC-β3(N260G), or PLC-β3(N260S) [all designated as PLC-β3(N260*)] as described in (41). Data are plotted as percent of maximal response obtained with PLC-β3. Data are mean ± SEM of three experiments. (B) Deficiency in termination of Gαq-stimulated PLC activity of a GAP-deficient mutant of PLC-β3. PLC activity was quantified with [3H]PtdIns(4,5)P2-containing phospholipid vesicles reconstituted with purified P2Y1 receptor, Gαq, and Gβ1γ2. Vesicles were incubated with 300 nM PLC-β3 or PLC-β3(δEF) in the absence (open circles) or presence of the P2Y1 receptor agonist 2MeSADP (300 nM; black squares) and either 30 μM GTP or 100 nM GTPγS for 90 s before addition of P2Y1 receptor antagonist MRS2500 (50 μM; red squares) or vehicle. Incubations were continued for an additional 165 s. Data are plotted as percent of the maximal response observed with either PLC-β3 or PLC-β3(δEF) in the presence of agonist plus GTPγS. (C) Delayed termination of the photoresponse in Drosophila expressing a GAP-deficient mutant of PLC-β. Electro-retinograms from flies harboring wild-type PLC-β (NORPA, blue) or a mutant form (NORPAN262A, red) deficient in capacity to accelerate the GTPase activity of Gαq. Flies ~1 day posteclosion were dark-adapted for 2 min before exposure to 5-s pulses of orange light indicated by the event marker below each electroretinogram. At right are plotted deactivation rates and maximal amplitudes for the average of ten individual electroretinograms. Error bars indicate SEM. Expression of the norpA transgenes was confirmed by immunoblot (gel) of head extracts prepared from flies ~1 day posteclosion. (D) GAP activity of a mutant of PLC-β found in pancreatic cancer. PLC-β3 was mutated at a position (R258) (21) equivalent to a homozygous substitution identified in PLC-β4 during genome-wide profiling of pancreatic cancers (30). GAP activity of PLC-β3(R258Q) was compared with that of PLC-β3 as described in (A) above. Data are mean ± SEM of three experiments.

Gary L. Waldo, et al. Science. ;330(6006):974-980.
4.
Fig. 1

Fig. 1. From: Kinetic Scaffolding Mediated by a Phospholipase C-? and Gq Signaling Complex.

Structure of Gαq•PLC-β3. (A) Domain architecture of PLC-β3 drawn to scale and consisting of a N-terminal PH domain, a series of four EF hands, a catalytic TIM barrel, a C2 domain, and a carboxy-terminal (CT) domain. The CT domain is not necessary for Gαq binding (fig. S2), and, therefore, PLC-β3 truncated at residue 886 was used to facilitate crystallization. Three distinct regions of PLC-β3 that interact with Gq are indicated by red numerals. (B) Overall structure of the AlF4-dependent complex of Gαq•PLC-β3 as viewed from the plane of the membrane. PLC-β3 is depicted as a ribbon cartoon with domains colored as in (A). Activated Gαq is depicted as a green surface with nucleotide-dependent switches (Sw1 to Sw3) in shades of red. Hα1/Hα2 (red 3) at the end of the C2 domain of PLC-β3 lies within the canonical effector-binding region of Gαq formed by α3 starting at M248 (21), the subsequent loop containing W263, and switch 2 containing Q209. The X/Y linker (orange) connects the two halves of the catalytic TIM barrel, and an ordered portion of the linker occludes the active site of the lipase highlighted by the Ca2+ (yellow ball) cofactor. (C) Surfaces of Gαq highlighting switches (top) in comparison to regions (lower images) of Gα subunits that interact with PLC-β3, other effectors, and RGS proteins. Interactions involving the EF3/4 loop are yellow except for overlap (dark blue) involving other regions of PLC-β3 (light blue). Gα subunits use a common interface (green) to engage four distinct effectors, and a different interface engages seven distinct RGS proteins (dark purple). Details of the analyses are supplied in (41). (D) Model for activation of PLC-β3 by GTP-activated Gαq. Gαq (green) bound to GDP is sequestered by Gβγ (red and yellow) and does not interact with PLC-β, depicted as a gold toroid except for its CT domain (light pink) and X/Y linker (orange cylinder and dotted lines). The CT domain basally associates with membranes, whereas the X/Y linker blocks the lipase active site. Upon activation of heterotrimeric Gq, Gαq-GTP dissociates from Gβγ and interacts with the main portion of PLC-β. Complex formation anchors and orients the lipase active site at membranes, leading to repulsion of the X/Y linker and freeing the active site for hydrolysis of PtdIns(4,5)P2 into diacylglycerol (DAG) and Ins(1,4,5)P3 (IP3).

Gary L. Waldo, et al. Science. ;330(6006):974-980.
5.
Fig. 2

Fig. 2. From: Kinetic Scaffolding Mediated by a Phospholipase C-? and Gq Signaling Complex.

Structure of the effector binding interface of Gαq•PLC-β3. (A) Ribbon diagram of the interface between the Hα1/Hα2 region of PLC-β3 (blue) and the effector binding pocket of Gαq located between α3 (green) and Sw2 (pink). Interfacial residues (sticks) are labeled; hydrogen bonds are indicated by dashed lines. (B) Comparison of PLC sequences (21) at the end of the C2 domain (Cβ8) encompassing Hα1/Hα2. α helices (cylinders) and β sheets (arrows) were assigned by using crystal structures of PLC-β3 as reported here (PDB 3OHM), PLC-β2 (2ZKM), and PLC-δ1 (1DJX). C termini are indicated for full-length PLC-δ1 as well as the crystallized fragments of PLC-β2 and -β3. Residues in PLC-β3 that interact with Gαq are underlined in red. Dots indicate every 10th residue. (C) Comparison of effectors bound to Gα subunits. The major effector binding surface of Gαq (green with switches in red) engages Hα1/Hα2 (blue cylinders) of PLC-β3 through indicated residues (sticks) surrounding Pro862. Structurally analogous α helices (gray cylinders) and residues (blue sticks) in other effectors are highlighted after superimposition of bound Gα subunits (not shown). PDE-γ, cyclic GMP phosphodiesterase-γ. (D) Mutational analyses of Hα1/Hα2. PLC-β3 mutants harboring the indicated single substitutions were assessed for capacity to be activated upon cotransfection with Gαq in COS-7 cells as measured by [3H]inositol phosphate production. Further experimental details are described in figs. S3, A to C, and S4. (E) Requirement of Hα1/Hα2 for activation of PLC-β3 by Gαq assessed with purified proteins. [3H]PtdIns(4,5)P2-containing phospholipid vesicles reconstituted with purified P2Y1 receptor, Gαq, and Gβ1γ2 were used to assess the capacity of wild-type or PLC-β3 mutants (300 nM) to hydrolyze PtdIns(4,5)P2 in the absence (basal) or presence of a P2Y1 receptor agonist (2MeSADP, 1 μM) plus 100 nM GTPγS (agonist + GTPγS). Data are mean ± SEM from four independent experiments. (F) Grafting Hα1/Hα2 onto PLC-δ1 confers responsiveness to Gαq. Activities of purified proteins were compared as in (E). Residues 847 to 886 of PLC-β3 were added to the end of PLC-δ1 to create PLC-δ1(Hα1/Hα2); starred variant consists of PLC-δ1(Hα1/Hα2) with additional substitutions (D610R and N612D) of PLC-δ1 to analogous PLC-β3 residues (see domain architectures in fig. S7A).

Gary L. Waldo, et al. Science. ;330(6006):974-980.

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