Effects of GRM3 alterations on cell growth and MEK phosphorylation. (a) Somatic alterations in GRM3 cause increased proliferation in reduced serum. We seeded A375 pooled GRM3 clones expressing wild-type, p.Gly561Glu, p.Ser610Leu, p.Glu767Lys, p.Glu870Lys or vector alone in 96-well plates in the presence of reduced serum (1% FBS). We harvested the plates and analyzed them by SYBR Green I on a BMG Labtech FluorOptima. Error bars, standard deviation (s.d.). (b) Mutant GRM3 shows anchorage-independent growth. We seeded Mel-STR cells in a top plug of agar and allowed them to incubate for 2 weeks before analysis by light microscopy and counting using US National Institutes of Health (NIH) ImageJ software (see ). Error bars, s.d. n = 3. Students t-tests in all instances showed a *P < 0.05, except for in the test of vector versus wild type. (c) Mutant GRM3 activates MEK1/2 upon DCG-IV stimulation in Mel-STR and A375 cells. We serum starved Mel-STR pooled GRM3 clones seeded in 6-well dishes for 4 h before the addition of either 2.5 μM DCG-IV or vehicle for 10 min. After lysis of the cells, we analyzed the lysates on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and immunoblotted them with corresponding antibodies. We generated the ratios shown by ImageJ and Microsoft Excel analysis of phosphorylated protein to total protein blots. (d) Mutant GRM3 activates MEK1/2 upon DCG-IV stimulation in A375 cells. We analyzed A375 pooled GRM3 clones as described in c.

(a) A375 pooled GRM3 clones in the absence of stimulus migrate as well as those stimulated with the group 2 metabotropic agonist DCG-IV. We seeded A375 clones in Boyden chambers in either the absence of stimulus of, or in the presence of, 2.5 μM DCG-IV and assessed them for their ability to migrate 16 h later. Error bars, s.d. (b) We analyzed Mel-STR pooled GRM3 clones for migration as described in a. We analyzed stained wells using a Zeiss microscope 10× lens and counted them with NIH ImageJ software. Error bars, s.d. n = 3. (c) We intravenously injected NOD/SCID mice with A375 pooled GRM3 clones expressing the wild-type, p.Gly561Glu, p.Ser610Leu, p.Glu767Lys, p.Glu870Lys or vector alone and examined them after nine weeks. The graph indicates the number of mice that had lung macrometastases (n = 10; P < 0.05, Fisher’s exact test). Shown are representative images of lungs from mice injected with the vector, wild type or mutant-expressing A375 clones.

Expression of mutant GRM3 provides cell proliferation and survival signals in melanoma. (a) Our protein blot analysis shows that expressing GRM3 shRNA decreases endogenous GRM3 levels. We analyzed HEK 293T cells co-transfected with shRNA targeting GRM3 and FLAG-GRM3 by immunoblot. We analyzed the lysates in parallel using anti-GAPDH. (b,c) Our quantitative RT-PCR (qRT-PCR) analysis shows that GRM3 shRNA decreases endogenous levels of GRM3. Error bars, s.d. n = 3. qRT-PCR analysis of the wild-type GRM3 cell line (34T) (b) and the mutant (p.Gly561Glu) GRM3 cell line (36T) (c) using GRM3- or GAPDH-specific primers. (d–h) Growth curves of representative melanoma cell lines transduced with shRNA. Error bars, s.d. n = 4. (i–m) Stable knockdown of GRM3 in mutant-GRM3–expressing cells causes decreased migration compared to wild-type–expressing cells. We seeded wild-type GRM3 melanoma cell lines stably transduced (i,j) or mutant GRM3 melanoma cell lines stably transduced (k–m) with either empty vector, shRNA #1 or shRNA #3 (GRM3) in Boyden chamber wells in triplicate and incubated them for 16–72 h before analysis. We quantitated the results using a Student’s t-test. Error bars, s.d. n = 3. (n,o) We subcutaneously injected Nu/Nu mice with either 31T or 36T GRM3 clones stably infected with pLKO.1, shRNA #1 or shRNA #3 against endogeneous GRM3 or 31T (wild-type GRM3) or 63T (p.Glu573Lys) GRM3 clones stably infected with the doxycycline-inducible TRIPz NC, sh639 or sh742 shRNA against endogenous GRM3. Graphs show volumetric measurements of 31T (n), 36T GRM3 (constitutive knockdown clones) (o), 31T (doxycycline-inducible clones) (p) or 63T GRM3 (doxycycline-inducible clones) (q) tumor-bearing mice. We performed all in vivo studies from n = 6 mice. Error bars, s.d.

Melanoma cell lines expressing GRM3 mutants show increased sensitivity to inhibition of MEK by AZD-6244. (a) Immunoblot analysis of representative melanoma cell lines harboring either wild-type or mutant GRM3. We treated the cells with the indicated concentration of AZD-6244 and analyzed them for ERK1/2 activation. We treated cells for 1 h with AZD-6244 or vehicle alone as a control. We subjected the lysates to protein blot analysis with anti-ERK1/2 (α-ERK1/2), anti– P-ERK1/2 (α-P-ERK1/2) and anti-GAPDH as a loading controls. (b) Representative dose-response curves showing the efficacy of AZD-6244 against GRM3 mutant lines compared to wild-type GRM3 lines. The relative cell numbers after we treated the cells for 72 h with increasing concentrations (0.002–30 μM) of AZD-6244, as estimated by CellTiter-Glo and plotted as percent survival, as compared to vehicle-treated control, versus log [AZD-6244] concentration in nM (where 1 is 10 nM AZD-6244). We generated fitted lines using four-parameter nonlinear regression. Error bars, s.d. n = 3. (c) FACS analysis of wild-type (2T) and p.Ser154Phe, p.Asp280Asn, p.Arg352Trp or p.Glu870Lys mutant (68T) cells showing the cell-cycle distribution (propidium iodide staining; x axis) versus cell count (y axis). Shown are representative plots. (d) Quantification of FACS-sorted AZD-6244–treated cells. We determined the percentage of apoptotic cells based on the subG1 population for vehicle-treated cells or AZD-6244–treated cells. Error bars, s.d. (e) Immunoblot analysis of representative melanoma lines expressing wild-type or mutant GRM3 after AZD-6244 treatment using the indicated antibodies to assess PARP cleavage.