Activation of heterotrimeric G proteins by Smoothened

Abstract

The mechanisms by which the activation of Smoothened (Smo), a protein essential to the actions of the Hedgehog family of secreted proteins, is translated into signals that converge on the Gli transcription factors are not fully understood. The seven-transmembrane structure of Smo has long implied the utilization of heterotrimeric GTP-binding regulatory proteins (G proteins); however, evidence in this regard has been indirect and contradictory. In the current study we evaluated the capacity of mammalian Smo to couple to G proteins directly. We found that Smo, by virtue of what appears to be constitutive activity, activates all members of the G(i) family but does not activate members of the G(s), G(q), and G(12) families. The activation is suppressed by cyclopamine and other inhibitors of Hedgehog signaling and is enhanced by the Smo agonist purmorphamine. Activation of G(i) by Smo is essential in the activation of Gli in fibroblasts, because disruption of coupling to G(i) with pertussis toxin inhibits the activation of Gli by Sonic hedgehog and a constitutively active form of Smo (SmoM2). However, G(i) does not provide a sufficient signal because a truncated form of Smo, although capable of activating G(i), does not effect activation of Gli. Rescue of pertussis toxin-inhibited activation of Gli by Sonic hedgehog can be achieved with a constitutively active Galpha(i)-subunit. The data suggest that Smo is in fact the source of two signals relevant to the activation of Gli: one involving G(i) and the other involving events at Smo's C-tail independent of G(i).

Fig. 1.

Smo is coupled to G proteins of the G_i family. (A) Sf9 cells were infected with a mixture of baculoviruses encoding Gα-subunits (corresponding to the indicated G protein), Gβ₁, and Gγ₂, each at an MOI of 1, with (gray bars) or without (black bars) a baculovirus encoding Smo (MOI = 2). At 48 h, membranes were isolated, and [³⁵S]GTPγS binding was evaluated for the Gα-subunits as described in Materials and Methods. [³⁵S]GTPγS binding is expressed as fold stimulation of binding to each G protein alone. For reference, the maximal average cpm for each G protein were as follows: ≈500 (G_i1), ≈700 (G_i2), ≈50 (G_i3), ≈2,000 (G_o), ≈1,000 (G_z), ≈10,000 (G_q), ≈250 (G₁₂), ≈2,500 (G₁₃), and ≈1,000 (G_s). Shown beneath the graph is a series of Western blots for Gα-subunits in the Sf9 membranes (10 μg of membrane protein per lane) using individual Gα-specific antisera. (B) Sf9 cells were infected with baculoviruses encoding Gα_i2, Gβ₁, and Gγ₂, each at an MOI of 1, and Smo at MOIs ranging from 0 to 5. Membranes were isolated, and [³⁵S]GTPγS binding was evaluated; binding is expressed as cpm. (C) Sf9 cells were infected with baculoviruses encoding Smo (MOI = 2), Gα, Gβ₁, and Gγ₂. Membranes were incubated for 10 min with vehicle (black bars) or 5 μM cyclopamine (gray bars) before the addition of [³⁵S]GTPγS. [³⁵S]GTPγS binding of 1-fold corresponds to each G protein alone. (D) Dose-dependent inhibition of Smo-promoted [³⁵S]GTPγS binding to G_i2 was evaluated for cyclopamine (filled circles), KAAD-cyclopamine (open circles), SANT-1 (filled squares), and tomatidine (open square). Binding in the absence of any compound was set at 100%, and Smo-independent binding was set at 0%. Each value in the four graphs represents the average of three independent experiments performed in triplicate ± 1 SE.

Fig. 2.

Smo both activates and utilizes G_i in mammalian cells. (A) Mouse embryonic fibroblasts were treated for 45 min with vehicle (DMSO) or 10 μM purmorphamine in serum-free medium. Activation of G_i was determined in subsequently prepared membranes as described in Fig. 1 by using vehicle (white bars), 5 μM cyclopamine (black bars), or 10 μM purmorphamine (gray bars). (B) HEK 293 cells were transfected with empty pcDNA vector or SmoM2. After 48 h, membranes were prepared and used to determine the activation of G_i in the absence (white bars) or presence (black bars) of KAAD-cyclopamine. (C) NIH 3T3 cells were transiently transfected with expression vectors encoding Shh, oncogenic Smo (SmoM2), Gli1, Gli2, or empty pcDNA vector, together with reporters encoding Gli-luciferase and a control Renilla luciferase. After the cells reached confluency, the medium was replaced with 0.5% FCS (black bars) or 0.5% FCS plus 100 ng/ml PTX (gray bars). Luciferase activities were determined 24 h later. Results are expressed as the ratio of firefly/Renilla luciferase activities and represent an average of three independent experiments performed in triplicate ± 1 SE. (D) NIH 3T3 cells were transfected with an expression vector encoding Shh or with empty pcDNA vector. Cells treated subsequently with or without PTX were lysed after 24 h to extract total RNA. Semiquantitative RT-PCR was performed for Ptch-1 and β-actin. The result is representative of three independent experiments.

Fig. 3.

The C-tail of Smo is required for activation of Gli but not G_i. (A) Membranes from Sf9 cells infected with recombinant baculoviruses for Smo and a C-tail-deleted mutant of Smo (SmoΔC) were subjected to SDS/PAGE (10 μg of protein per lane) and then, after transfer to nitrocellulose, probed with antibodies directed against the C-terminal (H300) or N-terminal (N19) aspects of Smo. Solid arrowheads denote the position of full-length Smo (86 kDa), and the empty arrowhead denotes the position of truncated Smo (62 kDa). (B) [³⁵S]GTPγS binding was evaluated as described in Fig. 1 for two members of the G_i family, G_i1 and G_i2, expressed with (gray bars) or without (black bars) SmoΔC in Sf9 cells. Results shown are representative of three independent experiments performed in triplicate. (C) SmoΔC-stimulated binding of [³⁵S]GTPγS to the G proteins was evaluated in the presence or absence of 5 μM cyclopamine as described in Fig. 1 for Smo. (D) NIH 3T3 cells were transfected with empty vector (pcDNA), SmoM2, or a C-tail-deleted mutant of SmoM2 (SmoM2ΔC) together with Gli (and Renilla) luciferase reporters, then assayed for luciferase activities 24 h thereafter, as described in Fig. 2. (Inset) SmoM2 and SmoM2ΔC express equivalently in HEK 293 cells (Western blot with N-19); expression in 3T3 cells was not high enough for detection.

Fig. 4.

Partial reversal of PTX inhibition of Smo signaling by a constitutively active mutant of Gα_i. NIH 3T3 cells were cotransfected with the indicated combination of vectors and, after reaching confluency, were incubated in 0.5% FCS with or without 100 ng/ml PTX, as described in Fig. 2. Gli-dependent luciferase activity was determined 24 h later as described; activities for Shh, SmoM2, Shh plus Gα_i2Q205L, and SmoM2 plus Gα_i2Q205L without PTX were each set to 100%. Results are one of three equivalent experiments performed in triplicate.