Identification of differentially expressed Gpr116 (Adgrf5) transcript variants in mouse kidney

Abstract

Adhesion G protein-coupled receptors (aGPCRs) are important and understudied modulators of physiological processes. Unlike class A GPCRs that are activated by soluble ligands, aGPCRs are self-activated by a cryptic tethered agonist after holoreceptor dissociation. We recently demonstrated that kidney-specific ADGRF5/Gpr116 knockout causes luminal membrane accumulation of V-ATPase in acid-secreting A-type intercalated cells (AICs) in the collecting ducts and a significant reduction in urine pH (Zaidman et al. 2020). Previous work suggests that aGPCRs, and Gpr116 in particular, undergo significant tissue-specific mRNA processing that results in holoreceptors with unique and variable N-terminal structures (Knierim et al. 2019). These aGPCR splice variants possess singular structure-function relationships that are integral to endogenous self-activation. Therefore, identifying tissue-specific transcript variants is critical to understanding the physiological roles of aGPCRs. Besides robust expression in AICs, Gpr116 is also transcribed in endothelial cells (ECs) in the kidney. We hypothesized that unique Gpr116 transcript variants are expressed in renal AICs and ECs. To test this hypothesis, we enzymatically digested kidneys from Gpr116flag V1B1eGFP mice and enriched for ICs (CD31−, GFP+) and ECs (CD31+, GFP−) by FACS. Gpr116 was not detected in CD31− GFP− sorted cells. IC enrichment was confirmed by qPCR by detection of Atp6v0d2, c-kit (CD117), Slc4a1, and Slc4a9 transcripts. EC enrichment was confirmed by detection of Kdr and Nrp1 transcripts. Next, we assayed cDNA from these cell populations for exon variants by PCR, and subsequent nanopore sequencing. We detected and aligned three exons that undergo differential expression in the kidney: exons 2, 12, and 22. Both IC and EC populations were positive for all three differentially expressed exons, but also expressed Gpr116 transcripts without the variable regions, suggesting multiple Gpr116 isoforms in each cell type. However, EC markers were detected in GFP+ ICs, demonstrating some EC contamination in the sorted ICs. To test the tissue specificity of these differentially expressed exons, we assayed 7 other tissues from Gpr116flag V1B1eGFP mice. We detected exon 2 and 12 in lung, heart and fat; we did not detect it in pancreas, spleen, thymus or liver. We detected exon 22 in lung and fat; we did not detect it in pancreas, heart, spleen, thymus or liver. We detected Gpr116flag in the lung, heart, spleen, fat, thymus, kidney and liver, but not pancreas. In summary, we have identified three variable exons within the open reading frame of Gpr116. Future studies will characterize the roles of differentially expressed exons on the endogenous activation of Gpr116 and determine if transcript variants are cell-type specific. This study is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (4R00DK127215). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.