Figure 7. AMPK regulates the abundance of MGL-1 levels, while direct phosphorylation may affect MGL-1 function.

(A,B) Starvation reduces [MGL-1::GFP] levels significantly in the AIY [Pttx-3::RFP] of WT animals and to a lesser extent in aak-2 mutants (AIY neuron is shown in the circle). Increased [MGL-1::GFP] levels in starved aak-2 mutants can be reversed by targeted expression of aak-2 in the AIY interneurons using [Pttx-3::aak-2] transgene (n>15), Error bars represent ± SEM (two-way ANOVA ***p<0.0001). Scale bars are 20 μm. (C) Mutating the consensus AMPK phosphorylation site at serine 234 to a non-phosphorylable alanine in MGL-1 (S234A) resulted in a reduced forward locomotion rate that is not further reduced in aak-2; mgl-1 (S234A) double mutants (i) (n>10), (one-way ANOVA *p<0.05, **p<0.001). In the box and whisker plots the central line is the median, the edges of the box are the 25th and 75th percentiles, and the whiskers extend to the most extreme data points.

DOI: http://dx.doi.org/10.7554/eLife.16349.028

Figure 7—figure supplement 1. AMPK regulates MGL-1 by modulating its steady state mRNA levels while also affecting MGL-1 function through direct phosphorylation.

(A) [MGL-1::GFP] levels are unchanged in well fed WT and aak-2 mutants in the AIY interneurons (shown in the circle) indicated by [TTX-3::RFP]. Scale bars represent 20 μm. (B,C) Relative messenger RNA levels were analyzed with semi-quantitative RT-PCR in well-fed and starved animals. mgl-1 mRNA levels (normalized to WT) are significantly higher in starved aak-2 mutants compared to starved WT animals. Error bars represent ± SEM (two-way ANOVA *p<0.05). (D) A schematic showing the conserved AMPK phosphorylation site in C. elegans MGL-1 and human GRM2 and GRM3 group II metabotropic glutamate receptors. (E) Normalized [MGL-1::GFP] levels are unchanged upon mutating serine 234 in mgl-1 (n>10), Error bars represent ± SEM (one-way ANOVA ***p<0.0001).

DOI: http://dx.doi.org/10.7554/eLife.16349.030