Background: Patients with growth hormone (GH)-secreting pituitary tumors leading to early childhood-onset gigantism were recently found to harbor germline Xq26.3 microduplications including GPR101. GPR101 is an orphan G protein-coupled receptor (GPCR) that is highly expressed in the tumors of the patients. Little is known about GPR101. Methods: We characterized GPR101 transcripts in vitro in human tissues/cell lines by integrating 5’-RACE and RNAseq analysis, and we predicted the putative promoter region in silico. GPR101 expression was investigated at the mRNA and protein level in post-mortem human, rat, and zebrafish tissues, by qPCR, whole-mount in situ hybridization, and immunostaining. GPR101 signaling was studied in HEK293 and rat GH-secreting (GH3) cells using Glo sensor cAMP assay, luciferase reporter assays, and fluorescence resonance energy transfer (FRET) imaging. β-arrestin binding was assessed by luciferase complementation assay. Receptor localization in transfected cells was determined by confocal microscopy and FACS analysis. Results: Four GPR101 isoforms have been identified, characterized by different 5’ UTRs and a common 6.2 kb-long 3’UTR. Some of these isoforms were found to co-exist in the same cells. In silico analyses predicted a CpG-enriched promoter region within 1 kb upstream of the putative transcription start site. GPR101 was only expressed at low levels or unexpressed in almost all adult human tissues examined except for specific regions of the brain. High GPR101 expression was seen in human fetal pituitary starting at 19 weeks of gestation, but not in postnatal pituitary tissues. GPR101 expression was also seen in several areas of the brain during zebrafish and rat development. Signal transduction studies showed that GPR101 over-expression strongly activates the Gα-s/cAMP signaling pathway in basal conditions in both cell lines investigated, while effects on Gα-i and Gα-q-mediated pathways appeared to be minor. Three previously identified GPR101 variants (p.G31S, p.T293I, and p.E308D) did not differ significantly from wt using a FRET-based cAMP sensor. In transfected HEK293 cells, GPR101 was mainly localized to intracellular vesicles and constitutively coupled to β-arrestin 2. Conclusions: This study shows that different GPR101 transcripts exist, and that the brain is the major site of GPR101 expression across different species, suggesting that this GPCR plays an important role in brain/hypothalamic functions. GPR101 exhibits high basal constitutive activity by acting mainly through the Gα-s/cAMP pathway, for which the mitogenic effects are well established in pituitary GH-secreting cells. In addition, GPR101 is constitutively coupled to β-arrestin 2, which might explain its location in intracellular vesicles.
Characterization of GPR101: Transcripts Structure, Expression Pattern, and Signal Transduction Pathways
Trivellin G;
2016-01-01
Abstract
Background: Patients with growth hormone (GH)-secreting pituitary tumors leading to early childhood-onset gigantism were recently found to harbor germline Xq26.3 microduplications including GPR101. GPR101 is an orphan G protein-coupled receptor (GPCR) that is highly expressed in the tumors of the patients. Little is known about GPR101. Methods: We characterized GPR101 transcripts in vitro in human tissues/cell lines by integrating 5’-RACE and RNAseq analysis, and we predicted the putative promoter region in silico. GPR101 expression was investigated at the mRNA and protein level in post-mortem human, rat, and zebrafish tissues, by qPCR, whole-mount in situ hybridization, and immunostaining. GPR101 signaling was studied in HEK293 and rat GH-secreting (GH3) cells using Glo sensor cAMP assay, luciferase reporter assays, and fluorescence resonance energy transfer (FRET) imaging. β-arrestin binding was assessed by luciferase complementation assay. Receptor localization in transfected cells was determined by confocal microscopy and FACS analysis. Results: Four GPR101 isoforms have been identified, characterized by different 5’ UTRs and a common 6.2 kb-long 3’UTR. Some of these isoforms were found to co-exist in the same cells. In silico analyses predicted a CpG-enriched promoter region within 1 kb upstream of the putative transcription start site. GPR101 was only expressed at low levels or unexpressed in almost all adult human tissues examined except for specific regions of the brain. High GPR101 expression was seen in human fetal pituitary starting at 19 weeks of gestation, but not in postnatal pituitary tissues. GPR101 expression was also seen in several areas of the brain during zebrafish and rat development. Signal transduction studies showed that GPR101 over-expression strongly activates the Gα-s/cAMP signaling pathway in basal conditions in both cell lines investigated, while effects on Gα-i and Gα-q-mediated pathways appeared to be minor. Three previously identified GPR101 variants (p.G31S, p.T293I, and p.E308D) did not differ significantly from wt using a FRET-based cAMP sensor. In transfected HEK293 cells, GPR101 was mainly localized to intracellular vesicles and constitutively coupled to β-arrestin 2. Conclusions: This study shows that different GPR101 transcripts exist, and that the brain is the major site of GPR101 expression across different species, suggesting that this GPCR plays an important role in brain/hypothalamic functions. GPR101 exhibits high basal constitutive activity by acting mainly through the Gα-s/cAMP pathway, for which the mitogenic effects are well established in pituitary GH-secreting cells. In addition, GPR101 is constitutively coupled to β-arrestin 2, which might explain its location in intracellular vesicles.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
