β-Arrestin 1 Differentially Modulates cAMP and ERK Pathways Downstream of the FSH Receptor

Gonadotropins, including luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are essential regulators of reproductive function; however, the molecular characteristics and signaling properties of their recombinant forms in nonhuman primates remain incompletely defined. In this study, we performed a comparative sequence and functional analysis of the gonadotropin subunit cDNAs from Cynomolgus (Macaca fascicularis) and Rhesus monkeys (Macaca mulatta) and produced recombinant single-chain LHβ/α and FSHβ/α proteins for biochemical and signaling characterization. Sequence analysis revealed complete conservation of the α- and FSHβ subunit cDNAs between the two monkey species, whereas minor synonymous nucleotide variations were observed in the LHβ subunit. Recombinant FSHβ/α was expressed in CHO-K1 and CHO-S cells using dual epitope tagging, allowing for efficient detection and purification. Western blotting and enzymatic deglycosylation confirmed that both recombinant hormones were glycosylated, with N-linked glycosylation being the predominant modification. Functionally, recombinant monkey FSHβ/α stimulated dose-dependent cAMP accumulation in HEK293 cells expressing the human FSH receptor (hFSHR), demonstrating its full biological activity. cAMP production was significantly enhanced in β-arrestin 1 knockout cells, indicating that β-arrestin 1 acts as a negative regulator of FSH receptor (FSHR)-mediated Gαs signaling. In contrast, phosphorylated ERK1/2 (pERK1/2) requires β-arrestin 1, as knockout of this protein abolishes ERK activation. Pharmacological inhibition experiments further revealed that FSH-induced pERK1/2 activation largely depended on the cAMP/PKA pathway, whereas PKC inhibition had a minimal effect. Comparative analyses of cells expressing hFSHR or rat FSHR revealed distinct temporal patterns of pERK1/2 activation, reflecting species-specific signaling kinetics. Together, these results establish a robust system for the production of biologically active recombinant monkey gonadotropins and delineate the dual roles of β-arrestin 1 in modulating FSHR-mediated cAMP and ERK signaling pathways. This study provides new insights into the structure–function relationships of primate gonadotropins and β-arrestin–dependent GPCR regulation.