Regulation of the G-protein regulatory-Gαi signaling complex by nonreceptor guanine nucleotide exchange factors

Abstract

Group II activators of G-protein signaling (AGS) serve as binding partners for Gα(i/o/t) via one or more G-protein regulatory (GPR) motifs. GPR-Gα signaling modules may be differentially regulated by cell surface receptors or by different nonreceptor guanine nucleotide exchange factors. We determined the effect of the nonreceptor guanine nucleotide exchange factors AGS1, GIV/Girdin, and Ric-8A on the interaction of two distinct GPR proteins, AGS3 and AGS4, with Gα(il) in the intact cell by bioluminescence resonance energy transfer (BRET) in human embryonic kidney 293 cells. AGS3-Rluc-Gα(i1)-YFP and AGS4-Rluc-Gα(i1)-YFP BRET were regulated by Ric-8A but not by Gα-interacting vesicle-associated protein (GIV) or AGS1. The Ric-8A regulation was biphasic and dependent upon the amount of Ric-8A and Gα(i1)-YFP. The inhibitory regulation of GPR-Gα(i1) BRET by Ric-8A was blocked by pertussis toxin. The enhancement of GPR-Gα(i1) BRET observed with Ric-8A was further augmented by pertussis toxin treatment. The regulation of GPR-Gα(i) interaction by Ric-8A was not altered by RGS4. AGS3-Rluc-Gα(i1)-YFP and AGS4-Rluc-G-Gα(i1)-YFP BRET were observed in both pellet and supernatant subcellular fractions and were regulated by Ric-8A in both fractions. The regulation of the GPR-Gα(i1) complex by Ric-8A, as well as the ability of Ric-8A to restore Gα expression in Ric8A(-/-) mouse embryonic stem cells, involved two helical domains at the carboxyl terminus of Ric-8A. These data indicate a dynamic interaction between GPR proteins, Gα(i1) and Ric-8A, in the cell that influences subcellular localization of the three proteins and regulates complex formation.

FIGURE 1.

Regulation of AGS3-Rluc-Gα_i1-YFP and AGS4-Rluc-Gα_i1-YFP BRET by nonreceptor GEFs. AGS3-Rluc (10 ng) (A) and AGS4-Rluc (2 ng) (B) were expressed together with Gα_i1-YFP (250 ng) in HEK cells, and BRET was measured as described under “Experimental Procedures.” Ric-8A, AGS1, or GIV(1660–1870) was expressed as indicated. Data are presented as the mean ± S.E. from four experiments with triplicate determinations. *, p < 0.05 compared with control. The relative fluorescent units and relative luciferase units for sample points in A and B are presented in Table 1. Lower panels, Ric-8A, AGS1, and GIV(1660–1870) immunoblots. Ric-8A and AGS1 proteins were detected with affinity-purified anti-Ric-8A and anti-AGS1 antibody, respectively. GIV(1660–1870) was detected with anti-His₆ antibody. Each lane contains 50 μg of total protein, and the immunoblot is representative of three separate experiments. The GIV construct (pcDNA3.1/His::GIV(1660–1870)) encoded the carboxyl-terminal region of the protein as described under “Experimental Procedures.”

FIGURE 2.

Regulation of AGS3-Rluc-Gα_i1-YFP and AGS4-Rluc-Gα_i1-YFP BRET by Ric-8A. A, BRET data presented in A were extracted from the larger, complete datasets in B and C. Lower panels, 1% Nonidet P-40 lysates from HEK cells expressing AGS3-Rluc, AGS4-Rluc, and Gα_i1-YFP (250 ng of plasmid) and increasing amounts of Ric-8A as in the upper panel were subjected to SDS-PAGE and immunoblotting with Ric-8A antisera. The immunoblot is representative of two similar experiments. The numbers to the right of the immunoblots correspond to the migration of prestained Bio-Rad protein standards. *, p < 0.05 compared with their control. For AGS3-Rluc (10 ng) (B) and AGS4-Rluc (2 ng) (C), increasing amounts of Gα_i1-YFP (25–500 ng) and Ric-8A (as indicated in the figure) were expressed in HEK cells, and BRET was measured as described under “Experimental Procedures.” In some experiments, cells were pretreated with pertussis toxin (100 ng/ml) for 16 h. Data in B and C are presented as the mean ± S.E. from four to five experiments with triplicate determinations. *, p < 0.05 compared with their control. Relative fluorescence units (RFU) were measured for each sample in B and C as described under “Experimental Procedures.” Insets in B and C, data are presented as the percentage of control values (cells expressing only AGS3-Rluc and Gα_i1-YFP). Results are expressed as the mean ± S.E. of four to five (B) or three (C) independent experiments with triplicate determinations. Inset for B, RFU values for control cells transfected with 25, 50, 100, 250, and 500 ng of pcDNA3::Gα_i1-YFP were 41,729 ± 793, 65,720 ± 2,291, 98,810 ± 2,716, 150,534 ± 6,153, and 178,964 ± 5,871, respectively, and for PT-treated cells were 41,673 ± 2,445, 55,531 ± 3,899, 94,584 ± 6,931, 148,609 ± 5,501 and 160,708 ± 5,583, respectively. RFU values at each level of transfected pcDNA3::Gα_i1-YFP, with or without PT, were significantly different (p < 0.05) from the corresponding control value with the exception of the RFU value for pcDNA3::Gα_i1-YFP (25 ng), pcDNA3::Ric-8A (200 ng). C, RFU values for control cells transfected with 25, 50, 100, 250, and 500 ng of pcDNA3::Gα_i1-YFP were 37,816 ± 1559, 42,803 ± 1,628, 55,727 ± 1,858, 75,420 ± 2,836 and 93,555 ± 5,158, respectively, and for PT-treated cells were 40,313 ± 2,031, 47,200 ± 3,208, 62,215 ± 4,841, 83,990 ± 6,741, and 111,044 ± 7,388, respectively. *, p < 0.05 compared with their control. RFU values at each level of transfected pcDNA3::Gα_i1-YFP for PT-treated cells were significantly different (p < 0.05) from the corresponding control value with the exception of the RFU values the following samples: pcDNA3::Gα_i1-YFP (25 ng), pcDNA3::Ric-8A (100, 750, and 1,000 ng); pcDNA3::Gα_i1-YFP (50, 100, 250, and 500 ng), pcDNA3::Ric-8A (100 ng); pcDNA3::Gα_i1-YFP (500 ng), pcDNA3::Ric-8A (200 ng).

FIGURE 3.

Regulation of AGS3-Rluc-Gα_i1-YFP BRET by Ric-8A in the neuronal catecholaminergic cell line (CAD). AGS3-Rluc (10 ng) was expressed together with Gα_i1-YFP and Ric-8A as indicated, and BRET was measured as described under “Experimental Procedures.” Data are presented as the mean ± S.E. from three experiments with triplicate determinations. *, p < 0.05 compared with their control. Inset, data are presented as the percentage of control values (cells expressing only AGS3-Rluc and Gα_i1-YFP). Results are expressed as the mean ± S.E. of four independent experiments with triplicate determinations. RFU values for control cells transfected with 10 ng of phRluc::AGS3 and 50, 100, 250, and 500 ng of pcDNA3::Gα_i1-YFP were 21,969 ± 2,231, 24,097 ± 1,598, 35,383 ± 2,472, and 49,359 ± 4,926, respectively. RFU values at each level of transfected pcDNA3::Gα_i1-YFP were significantly different (p < 0.05) from the corresponding control value with the exception of the RFU values for pcDNA3-Gα_i1-YFP (50 ng), pcDNA3::Ric-8A (200 and 500 ng).

FIGURE 4.

Effect of RGS4 on AGS3-Rluc-Gα_i1-YFP and on AGS4-Rluc-Gα_i1-YFP BRET. AGS3-Rluc (10 ng) (left panel) and AGS4-Rluc (2 ng) (right panel) were expressed in HEK cells with Gα_i1-YFP (250 ng) in the presence and absence of Ric-8A, and BRET was measured as described under “Experimental Procedures.” RGS4-C2S (500 ng) was co-expressed as indicated. Data are presented as the mean ± S.E. from four experiments with triplicate determinations. *, p < 0.05 compared with their control. Lower panel, Ric-8A and RGS4 immunoblot. Each lane contains 50 μg of total protein, and the immunoblot is representative of two separate experiments.

FIGURE 5.

Ric-8A regulates AGS3-Rluc-Gα_i1-YFP and AGS4-Rluc-Gα_i1-YFP BRET in both pellet and supernatant fractions. A, AGS3-Rluc (10 ng) and AGS4-Rluc (2 ng) were expressed in HEK cells with Gα_i1-YFP (250 ng), and BRET was measured in intact cells, pellet, and supernatant as described under “Experimental Procedures.” Ric-8A was expressed as indicated. Results are expressed as the mean ± S.E. of three independent experiments with triplicate determinations. *, p < 0.05 compared with their control. B, AGS3-Rluc (10 ng) was expressed in the neuronal catecholaminergic cell line (CAD) together with Gα_i1-YFP (50 ng) and Ric-8A as indicated, and BRET was measured as described under “Experimental Procedures.” Data are presented as the mean ± S.E. from three experiments with triplicate determinations. *, p < 0.05 compared with their control as determined by Student's t test. The relative fluorescent units and relative luciferase units for sample points in A and B are presented in Table 2. C, AGS3-Rluc (10 ng) and Gα_i1-YFP (50 ng) (left panel) or Gα_i1-YFP (250 ng) (right panel) in the presence and absence of Ric-8A (1,000 ng) were expressed in HEK cells, and BRET was measured in intact cells, pellet, and supernatant fractions prepared from control cells or cells pretreated with PT as described under “Experimental Procedures.” RFU and RLU are presented as the percentage of control values obtained in cells expressing only AGS3-Rluc and Gα_i1-YFP. Results are expressed as the mean ± S.E. of two independent experiments with triplicate determinations. *, p < 0.05 Ric-8A versus control. **, p < 0.05 PT versus non-PT treated control. Basal levels of RFU and RLU for control cells were as follows: 50 ng of pcDNA3::Gα_i1-YFP: intact cell RFU = 89,412 ± 837, RLU = 524,526 ± 115,170; pellet RFU = 61,037 ± 9,021, RLU = 182,237 ± 82,483; supernatant RFU = 28,810 ± 7,388, RLU = 455,064 ± 255,751. 250 ng of pcDNA3::Gα_i1-YFP: intact cell RFU = 141,573 ± 6,692, RLU = 338,131 ± 75,783; pellet RFU = 113,476 ± 11,240, RLU = 204,852 ± 76,241; supernatant RFU = 41,276 ± 9,572, RLU = 204,829 ± 110,133.

FIGURE 6.

Effect of carboxyl-terminal truncated Ric-8A on Gα expression. A, complementation assay in Ric-8A^−/− murine embryonic stem cells. Ric-8A^−/− ES cell clones stably expressing full-length Ric-8A, Ric-8A (Met¹–Asn⁴⁹²), Ric-8A (Met¹–Asn⁴⁵³), or pcDNA3.1 Hygro were lysed and fractionated into pellet and supernatant as described elsewhere (66). Equal protein from the fractions was immunoblotted (IB) for Ric-8A, Gα_i1/2 (B084), and Gα_q/11 (Z811). B, AGS3-Rluc (10 ng), Gα_i1-YFP (500 ng), and Ric-8A WT, Ric-8A (Met¹–Asn⁴⁵³), and Ric-8A (Met¹–Asn⁴⁹²) (1000 ng) in pcDNA3 were expressed in HEK cells, and relative fluorescence units (RFU) were measured as described under “Experimental Procedures.” Results are expressed as the mean ± S.E. of four independent experiments with triplicate determinations. *, p < 0.05 compared with control.

FIGURE 7.

Effect of truncated Ric-8A on AGS3-Rluc-Gα_i1-YFP BRET. A, BRET data presented in A were extracted from the larger, complete datasets in B. *, p < 0.05, compared with its control. Lower panel, Ric-8A immunoblot. Each lane contains 50 μg of total protein, and the immunoblot presented is representative of three separate experiments. The numbers to the right of the immunoblots correspond to the migration of prestained Bio-Rad protein standards. B, AGS3-Rluc (10 ng), increasing amounts of Gα_i1-YFP (25–500 ng) and Ric-8A WT, Ric-8A (Met¹–Asn⁴⁵³), and Ric-8A (Met¹–Asn⁴⁹²) (as indicated in the figures) were expressed in HEK cells, and BRET signals were measured as described under “Experimental Procedures.” Middle panel, RFUs for each sample are presented as the percentage of control values (cells expressing only AGS3-Rluc and Gα_i1-YFP). RFU values for control cells transfected with 25, 50, 100, 250, and 500 ng of pcDNA3::Gα_i1-YFP 50,476 ± 6,117, 62,489 ± 6,986, 87,708 ± 11,705, 120,783 ± 16,267, and 155,140 ± 20,720, respectively. Lower panel, RLU are presented as the percentage of control values obtained in cells expressing only AGS3-Rluc and Gα_i1-YFP. Relative luminescence unit values for control cells transfected with 25, 50, 100, 250, and 500 ng of pcDNA3::Gα_i1-YFP were 462,452 ± 134,067, 456,049 ± 110,347, 400,833 ± 100,309, 263,295 ± 52,793, and 207,460 ± 45,087, respectively. Results are expressed as the mean ± S.E. of four independent experiments with triplicate determinations. *, p < 0.05 compared with their control. C, effect of PT pretreatment on the regulation of AGS3-Rluc-Gα_i1-YFP BRET by carboxyl-terminal truncated Ric-8A. AGS3-Rluc (10 ng) and Gα_i1-YFP (50 ng) (left panel) or Gα_i1-YFP (250 ng) (right panel) were expressed in HEK cells and BRET signals, RFU and RLU values were measured as described under “Experimental Procedures.” The relative fluorescent units and relative luciferase units are presented in Table 3. Ric-8A and truncated Ric-8A mutants were expressed as indicated. In some experiments, cells were pretreated with pertussis toxin (100 ng/ml) for 16 h. Results are expressed as the mean ± S.E. of three independent experiments with triplicate determinations. *, p < 0.05 compared with their control.