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The lysophosphatidic acid receptor-3 (LPAR3) is a G protein-coupled receptor that mediates viability among malignant cells and aggressiveness among certain tumors. The study's objective was to determine the interplay between LPAR3 and miRNAs to impact key cellular signaling pathways. Using SK-Mel-2 and SK-Mel-5 melanoma cells, wild-type and mutated receptors were stably expressed to explore molecular mechanisms. LPAR3 signaling induced miR-122-5p intracellularly and subsequently its inclusion into exosomes. This amplification resulted in less abundant Wnt1, maintenance of GSK3 inactivation and to a lesser extent, partial degradation of b-catenin. The surge in miR-122-5p and reduction in Wnt1 originated from signaling at the Src homology 3 (SH3) ligand-binding motif within the third intracellular loop of LPAR3, because mutant receptors did not increase miR-122-5p and had a weakened capacity to reduce Wnt1. In addition, a key mediator of melanoma survival signaling, the peroxisome proliferator-activated receptor gamma coactivator 1-a (PPARGC1A/PGC1), was involved in miR-122-5p transcription. In conclusion, this study highlights the powerful role miRNAs have in fine-tuning specific G protein-coupled receptor-mediated signaling events by altering the transcription of signaling transduction pathway components. This study also identifies that LPAR3 increases miR-122-5p expression, which occurs mechanistically through the SH3 domain and helps explain why miR-122-5p increases are detected in cancer patient serum.Implications: LPAR3 is partially responsible for the production and secretion of miR-122-5p, found in the serum of a wide variety of patients with cancer.

<p>Supplementary Figure 5. miR-122-5p abundance fluctuates in heart tissue, resultant from LPAR3 expression. Tissues were collected from knock-out mice lacking the first exon of the LPAR3 and their wild-type siblings and assessed for miR-122-5p and LPAR3. Heart tissue samples exhibited statistically lower miR-122-5p levels when compared to wild-type mice (*p<0.05). No significant differences were detected in liver and kidney tissue samples.</p>

<p>Supplementary Figure 4. Comparison of exogenously-added reagents. The qRT-PCR results display the relative abundance of miR-122-5p with no treatment (untreated) versus DharmaFECT alone, miR-122-5p mimic and miR-122-5p antagomir. The latter shows an unexpected increase, precluding its use.</p>

<p>Supplementary Figure 3. Microfluidics visualization of nanoparticles from LPAR3. The movies illustrate the difference between the smaller/almost indiscernible white dots, which are exosome nanoparticles, secreted by cells expressing LPAR3-wt versus the larger particles, likely multi-vesicular bodies, but not exosomes.</p>

<div>Abstract<p>The lysophosphatidic acid receptor-3 (LPAR3) is a G protein–coupled receptor that mediates viability among malignant cells and aggressiveness among certain tumors. The study's objective was to determine the interplay between LPAR3 and miRNAs to impact key cellular signaling pathways. Using SK-Mel-2 and SK-Mel-5 melanoma cells, wild-type and mutated receptors were stably expressed to explore molecular mechanisms. LPAR3 signaling induced miR-122-5p intracellularly and subsequently its inclusion into exosomes. This amplification resulted in less abundant Wnt1, maintenance of GSK3 inactivation and to a lesser extent, partial degradation of β-catenin. The surge in miR-122-5p and reduction in Wnt1 originated from signaling at the Src homology 3 (SH3) ligand–binding motif within the third intracellular loop of LPAR3, because mutant receptors did not increase miR-122-5p and had a weakened capacity to reduce Wnt1. In addition, a key mediator of melanoma survival signaling, the peroxisome proliferator-activated receptor gamma coactivator 1-α (PPARGC1A/PGC1), was involved in miR-122-5p transcription. In conclusion, this study highlights the powerful role miRNAs have in fine-tuning specific G protein–coupled receptor-mediated signaling events by altering the transcription of signaling transduction pathway components. This study also identifies that LPAR3 increases miR-122-5p expression, which occurs mechanistically through the SH3 domain and helps explain why miR-122-5p increases are detected in cancer patient serum.</p>Implications:<p>LPAR3 is partially responsible for the production and secretion of miR-122-5p, found in the serum of a wide variety of patients with cancer.</p></div>

<p>Supplementary Figure 2. Messenger RNA expression differences between cell types. Other mRNAs were assessed to compare relative LPAR3 expression as well as CCNG1.</p>