Identification of pathway-biased and deleterious melatonin receptor mutants in autism spectrum disorders and in the general population

Abstract

Melatonin is a powerful antioxidant and a synchronizer of many physiological processes. Alteration of the melatonin pathway has been reported in circadian disorders, diabetes and autism spectrum disorders (ASD). However, very little is known about the genetic variability of melatonin receptors in humans. Here, we sequenced the melatonin receptor MTNR1A and MTNR1B, genes coding for MT1 and MT2 receptors, respectively, in a large panel of 941 individuals including 295 patients with ASD, 362 controls and 284 individuals from different ethnic backgrounds. We also sequenced GPR50, coding for the orphan melatonin-related receptor GPR50 in patients and controls. We identified six non-synonymous mutations for MTNR1A and ten for MTNR1B. The majority of these variations altered receptor function. Particularly interesting mutants are MT1-I49N, which is devoid of any melatonin binding and cell surface expression, and MT1-G166E and MT1-I212T, which showed severely impaired cell surface expression. Of note, several mutants possessed pathway-selective signaling properties, some preferentially inhibiting the adenylyl cyclase pathway, others preferentially activating the MAPK pathway. The prevalence of these deleterious mutations in cases and controls indicates that they do not represent major risk factor for ASD (MTNR1A case 3.6% vs controls 4.4%; MTNR1B case 4.7% vs 3% controls). Concerning GPR50, we detected a significant association between ASD and two variations, Delta502-505 and T532A, in affected males, but it did not hold up after Bonferonni correction for multiple testing. Our results represent the first functional ascertainment of melatonin receptors in humans and constitute a basis for future structure-function studies and for interpreting genetic data on the melatonin pathway in patients.

Figure 1. Detection of MT₁ and MT₂ mutants by SDS-PAGE.

Lysates from HEK 293 cells transiently expressing the indicated receptors were separated by SDS-PAGE and analysis performed by Western blot using anti-Flag or anti-MT₁ (MT₁) (A) or anti-Myc or anti-MT₂ antibodies (MT₂) (B). Similar results were obtained in three additional experiments.

Figure 2. A. Sub-cellular localization of MT₁ mutants.

COS cells transiently expressing the indicated receptors were permeabilized or not with triton X-100 and total and surface exposed receptors detected by immunofluorescence microcopy with anti-Flag (MT₁) antibodies. B. Sub-cellular localization of MT2 mutants. COS cells transiently expression the indicated receptors were permeabilized or not with triton X-100 and total and surface exposed receptors detected by immunofluorescence microcopy with anti-myc (MT₂) antibodies. Similar results were obtained in three additional experiments.

Figure 3. Signaling of MT₁ mutants through the cAMP and ERK pathways.

HEK 293 cells were transiently (A, B) or stably (C–F) transfected with the indicated receptors. Inhibition of the cAMP pathway was measured by stimulating cells with forskolin alone (10 µM) or with forskolin and (A) 10 nM or (C) increasing concentration of melatonin for 60 min. Cyclic AMP levels were determined as described in Materials and Methods. ERK activation was measured by incubating cells with 100 nM melatonin for the indicated times (C–F). Phospho-ERK and ERK levels were determined as described in Materials and Methods. Data are means ± S.E.M. of three independent experiments each performed in duplicate (*, P<0.05; **, P<0.01; ***, P<0.001 vs wt).

Figure 4. Signaling of MT₂ mutants through the cAMP and ERK pathways.

HEK 293 cells were transiently (A, B) or stably (C–E) transfected with the indicated receptors. Inhibition of the cAMP pathway was measured by stimulating cells with forskolin alone (10 µM) or with forskolin and (A) 10 nM or (C) increasing concentration of melatonin for 60 min. Cyclic AMP levels were determined as described in Materials and Methods. ERK activation was measured by incubating cells with 100 nM melatonin for the indicated times (C–E). Phospho-ERK and ERK levels were determined as described in Materials and Methods. Data are means ± S.E.M. of three independent experiments each performed in duplicate (*, P<0.05; **, P<0.01; ***, P<0.001 vs wt).

Figure 5. Topology of MT₁ and MT₂ receptors.

The amino acid sequence of MT₁ (A) and MT₂ (B) receptors are shown and amino acids identified in receptor variants are highlighted in black (ℓ). Amino acids known to be involved in ¹²⁵I-MLT binding are circled in black. The NRY motif is highlighted in grey (C-ter, carboxyl terminal domain; e1, e2, e3, extracellular loops 1–3; i1, i2, i3, intracellular loops 1–3; N-ter, amino terminal domain).