The N-Linked Glycosylation Asn191 and Asn199 Sites Are Controlled Differently Between PKA Signal Transduction and pEKR1/2 Activity in Equine Follicle-Stimulating Hormone Receptor
Equine follicle-stimulating hormone receptor (eFSHR) contains four extracellular N-linked glycosylation sites, which play important roles in agonist-induced signal transduction. Glycosylation regulates G protein-coupled receptor mechanisms by influencing folding, ligand binding, signaling, trafficki...
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2025-03-01
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| author | Sung-Hoon Kim Munkhzaya Byambaragchaa Sei Hyen Park Myung-Hum Park Myung-Hwa Kang Kwan-Sik Min |
| author_facet | Sung-Hoon Kim Munkhzaya Byambaragchaa Sei Hyen Park Myung-Hum Park Myung-Hwa Kang Kwan-Sik Min |
| author_sort | Sung-Hoon Kim |
| collection | DOAJ |
| description | Equine follicle-stimulating hormone receptor (eFSHR) contains four extracellular N-linked glycosylation sites, which play important roles in agonist-induced signal transduction. Glycosylation regulates G protein-coupled receptor mechanisms by influencing folding, ligand binding, signaling, trafficking, and internalization. Here, we examined whether the glycosylated sites in eFSHR are necessary for cyclic adenosine monophosphate (cAMP) signal transduction and the phosphate extracellular signal-regulated kinase 1/2 (pERK1/2) response. We constructed mutants (N191Q, N199Q, N268Q, and N293Q) of the four N-linked glycosylation sites in eFSHR using site-directed mutagenesis. In wild-type (wt) eFSHR, the cAMP response gradually increased dose-dependently, displaying a strong response at the EC<sub>50</sub> and Rmax. Two mutants (N191Q and N199Q) considerably decreased the cAMP response. Both EC<sub>50</sub> values were approximately 0.46- and 0.44-fold compared to that of the eFSHR-wt, whereas Rmax levels were 0.29- and 0.45-fold compared to eFSHR-wt because of high-ligand treatment. Specifically, the EC<sub>50</sub> and Rmax values in the N268Q mutant were increased 1.23- and 1.46-fold, respectively, by eFSHR-wt. pERK1/2 activity in eFSHR-wt cells was rapid, peaked within 5 min, consistently sustained until 15 min, and then sharply decreased. pERK1/2 activity in the N191Q mutant showed a pattern similar to that of the wild type, despite impaired cAMP responsiveness. The N199Q mutant showed low pERK1/2 activity at 5 and 15 min. Interestingly, pERK1/2 activity in the N268Q and N298Q mutants was similar to that of eFSHR-wt at 5 min, but neither mutant showed any signaling at 15 min, despite displaying high cAMP responsiveness. Overall, eFSHR N-linked glycosylation sites can signal to pERK1/2 via PKA and the other signals, dependent on G protein coupling and β-arrestin-dependent recruitment. Our results provide strong evidence for a new paradigm in which cAMP signaling is not activated, yet pERK1/2 cascade remains strongly induced. |
| format | Article |
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| issn | 1467-3037 1467-3045 |
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| spelling | doaj-art-30738b94be9c43eb85cfbd90fb1a16d82025-08-20T02:11:16ZengMDPI AGCurrent Issues in Molecular Biology1467-30371467-30452025-03-0147316810.3390/cimb47030168The N-Linked Glycosylation Asn191 and Asn199 Sites Are Controlled Differently Between PKA Signal Transduction and pEKR1/2 Activity in Equine Follicle-Stimulating Hormone ReceptorSung-Hoon Kim0Munkhzaya Byambaragchaa1Sei Hyen Park2Myung-Hum Park3Myung-Hwa Kang4Kwan-Sik Min5Graduate School of Animal BioScience, Hankyong National University, Anseong 17579, Republic of KoreaCarbon-Neutral Resources Research Center, Hankyong National University, Aseong 17579, Republic of KoreaGraduate School of Animal BioScience, Hankyong National University, Anseong 17579, Republic of KoreaTNT Research, Sejong 30141, Republic of KoreaDepartment of Food Science and Nutrition, Hoseo University, Asan 31499, Republic of KoreaGraduate School of Animal BioScience, Hankyong National University, Anseong 17579, Republic of KoreaEquine follicle-stimulating hormone receptor (eFSHR) contains four extracellular N-linked glycosylation sites, which play important roles in agonist-induced signal transduction. Glycosylation regulates G protein-coupled receptor mechanisms by influencing folding, ligand binding, signaling, trafficking, and internalization. Here, we examined whether the glycosylated sites in eFSHR are necessary for cyclic adenosine monophosphate (cAMP) signal transduction and the phosphate extracellular signal-regulated kinase 1/2 (pERK1/2) response. We constructed mutants (N191Q, N199Q, N268Q, and N293Q) of the four N-linked glycosylation sites in eFSHR using site-directed mutagenesis. In wild-type (wt) eFSHR, the cAMP response gradually increased dose-dependently, displaying a strong response at the EC<sub>50</sub> and Rmax. Two mutants (N191Q and N199Q) considerably decreased the cAMP response. Both EC<sub>50</sub> values were approximately 0.46- and 0.44-fold compared to that of the eFSHR-wt, whereas Rmax levels were 0.29- and 0.45-fold compared to eFSHR-wt because of high-ligand treatment. Specifically, the EC<sub>50</sub> and Rmax values in the N268Q mutant were increased 1.23- and 1.46-fold, respectively, by eFSHR-wt. pERK1/2 activity in eFSHR-wt cells was rapid, peaked within 5 min, consistently sustained until 15 min, and then sharply decreased. pERK1/2 activity in the N191Q mutant showed a pattern similar to that of the wild type, despite impaired cAMP responsiveness. The N199Q mutant showed low pERK1/2 activity at 5 and 15 min. Interestingly, pERK1/2 activity in the N268Q and N298Q mutants was similar to that of eFSHR-wt at 5 min, but neither mutant showed any signaling at 15 min, despite displaying high cAMP responsiveness. Overall, eFSHR N-linked glycosylation sites can signal to pERK1/2 via PKA and the other signals, dependent on G protein coupling and β-arrestin-dependent recruitment. Our results provide strong evidence for a new paradigm in which cAMP signaling is not activated, yet pERK1/2 cascade remains strongly induced.https://www.mdpi.com/1467-3045/47/3/168equine FSHRN-glycosylation sitescAMP responsepERK1/2 activity |
| spellingShingle | Sung-Hoon Kim Munkhzaya Byambaragchaa Sei Hyen Park Myung-Hum Park Myung-Hwa Kang Kwan-Sik Min The N-Linked Glycosylation Asn191 and Asn199 Sites Are Controlled Differently Between PKA Signal Transduction and pEKR1/2 Activity in Equine Follicle-Stimulating Hormone Receptor Current Issues in Molecular Biology equine FSHR N-glycosylation sites cAMP response pERK1/2 activity |
| title | The N-Linked Glycosylation Asn191 and Asn199 Sites Are Controlled Differently Between PKA Signal Transduction and pEKR1/2 Activity in Equine Follicle-Stimulating Hormone Receptor |
| title_full | The N-Linked Glycosylation Asn191 and Asn199 Sites Are Controlled Differently Between PKA Signal Transduction and pEKR1/2 Activity in Equine Follicle-Stimulating Hormone Receptor |
| title_fullStr | The N-Linked Glycosylation Asn191 and Asn199 Sites Are Controlled Differently Between PKA Signal Transduction and pEKR1/2 Activity in Equine Follicle-Stimulating Hormone Receptor |
| title_full_unstemmed | The N-Linked Glycosylation Asn191 and Asn199 Sites Are Controlled Differently Between PKA Signal Transduction and pEKR1/2 Activity in Equine Follicle-Stimulating Hormone Receptor |
| title_short | The N-Linked Glycosylation Asn191 and Asn199 Sites Are Controlled Differently Between PKA Signal Transduction and pEKR1/2 Activity in Equine Follicle-Stimulating Hormone Receptor |
| title_sort | n linked glycosylation asn191 and asn199 sites are controlled differently between pka signal transduction and pekr1 2 activity in equine follicle stimulating hormone receptor |
| topic | equine FSHR N-glycosylation sites cAMP response pERK1/2 activity |
| url | https://www.mdpi.com/1467-3045/47/3/168 |
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