SET Protein Interacts with Intracellular Domains of the Gonadotropin-releasing Hormone Receptor and Differentially Regulates Receptor Signaling to cAMP and Calcium in Gonadotrope Cells

Abstract

Background: Mechanisms regulating signaling of mammalian GnRH receptor (GnRHR), which exhibits an atypical structure, are poorly known. Results: SET interacts with GnRHR, differentially impacts GnRHR coupling to calcium and cAMP signaling, and enhances GnRH stimulation of Gnrhr promoter activity. Conclusion:This represents the first identification of a GnRHR interacting partner that enhances its coupling to the cAMP pathway. Significance: SET is a novel regulator of GnRHR signaling.In mammals, the receptor of the neuropeptide gonadotropinreleasing hormone (GnRHR) is unique among the G proteincoupled receptor (GPCR) family because it lacks the carboxylterminal tail involved in GPCR desensitization. Therefore, mechanisms involved in the regulation of GnRHR signaling are currently poorly known. Here, using immunoprecipitation and GST pull-down experiments, we demonstrated that SET interacts with GnRHR and targets the first and third intracellular loops. We delineated, by site-directed mutagenesis, SET binding sites to the basic amino acids 66 KRKK 69 and 246 RK 247 , located next to sequences required for receptor signaling. The impact of SET on GnRHR signaling was assessed by decreasing endogenous expression of SET with siRNA in gonadotrope cells. Using cAMP and calcium biosensors in gonadotrope living cells, we showed that SET knockdown specifically decreases GnRHRmediated mobilization of intracellular cAMP, whereas it increases its intracellular calcium signaling. This suggests that SET influences signal transfer between GnRHR and G proteins to enhance GnRHR signaling to cAMP. Accordingly, complexing endogenous SET by introduction of the first intracellular loop of GnRHR in ␣T3-1 cells significantly reduced GnRHR activation of the cAMP pathway. Furthermore, decreasing SET expression prevented cAMP-mediated GnRH stimulation of Gnrhr promoter activity, highlighting a role of SET in gonadotropin-releasing hormone regulation of gene expression. In conclusion, we identified SET as the first direct interacting partner of mammalian GnRHR and showed that SET contributes to a switch of GnRHR signaling toward the cAMP pathway. G protein-coupled receptors (GPCR)3 represent the largest family of membranous receptors with more than 1000 members identified so far (1). GPCR process signals from a great diversity of endogenous and exogenous stimuli, including biogenic amines, peptides, glycoproteins, lipids, nucleotides, ions, proteases, photons, odors, and tastes. Because of their importance in a wide range of physiological and pathophysiological processes and the fact that they represent one of the most important drug targets, understanding the mechanisms regulating the efficacy and specificity of GPCR is a current challenge. GPCR-interacting proteins (GIP) have been shown to influence GPCR function (2). They are cytoplasmic proteins that bind to intracellular domains of GPCR and participate in the assembly of receptors into signal transduction complexes or "receptosomes." GIP influence signal transfer from the receptor to G ...

Alternative 3′ UTRs act as scaffolds to regulate membrane protein localization

“…3d, e and Extended Data Fig. 6 ) and others have shown that SET binds to positively charged amino acids in histone tails or cytoplasmic domains of membrane proteins 18 , 27 . According to the positive-inside rule for integral membrane proteins, the cytoplasmic domains of membrane proteins are enriched in positively charged amino acids for topological reasons 28 .…”

Proteomic Analysis of Brain Region and Sex-Specific Synaptic Protein Expression in the Adult Mouse Brain

“…Mutations in the gene encoding SET are linked to developmental delay and intellectual disabilities as well as to autosomal dominant 58 (MRD58), a form of mental retardation, characterized by significantly below average general intellectual functioning associated with delayed development, impairments in adaptive behavior, language delay and speech impairment [94][95][96]. Interestingly, SET interacts with intracellular domains of the gonadotropin-releasing hormone (GnRH) receptor and differentially regulates receptor signaling to cAMP and calcium in gonadotrope cells [97]. Notably, a recent study showed that SET expression is regulated by the neurohormone GnRH [98], providing a potential molecular basis for sex-specific differences in expression levels.…”

Overexpression of PP2A inhibitor SET oncoprotein is associated with tumor progression and poor prognosis in human non-small cell lung cancer

“…SET protein directly binds with PP2Ac through its both N-terminus and C-terminus regions, and inhibits PP2A phosphatase activity [ 20 ]. The SET oncoprotein participates in the regulation of a wide variety of molecular processes [ 21 - 24 ]. Of importance, SET plays an oncogenic role modulating signaling pathways with high relevance in human cancer [ 25 ].…”