Functions of GnRH receptors in GnRH neurons: *The G protein-coupled receptor (GPCR) systems have multimodular functions and provide cells with many possibilities to transduce incoming signals. A typical cell expresses several different GPCR genes, several combinations of G protein subunits, and multiple isoforms of the effector molecules that are activated by each type of G protein. The differential expression of these proteins allows modulation of signals at different levels, resulting in regulation that is characteristic for a specific cell type. The current understanding is that protein-protein interactions determine the mechanisms between the G proteins and particular GPCRs. However, several observations in specific cells and tissues have shown that different receptors coupling to the same G protein in a single cell, can elicit differential biochemical or cellular responses. The endogenous GnRH-Rs expressed in native and immortalized (GT1-7) GnRH neurons activate at least three G proteins, as indicated by the agonist-induced release of their specific alpha-subunits from the plasma membrane. Such coupling to the diverse array of G proteins provides GnRH neurons with several critical signaling pathways to transduce incoming signals. The differential expression of these regulatory proteins allows modulation of signals at many levels, resulting in messages that are optimal for specific cell functions. *Cellular distribution, receptor-receptor interactions, and signaling pathways of the murine GnRH-R tagged with Renilla luciferase (Rluc) and/or green fluorescent protein (GFP) were analyzed in GT1-7 GnRH neurons. The GFP-tagged GnRH-R was confined to a thin rim of cytosol at the plasma membrane, neuronal processes, and apparent synaptic junctions. GnRH stimulation caused redistribution of GnRH-R-GFP, with movement in close proximity to their bipolar extensions and increased intensity in synaptic connections. BRET2 assays revealed dose-dependent, GnRH-induced interaction between GFP-tagged GnRH-R and Rluc-tagged Galphaq, Galphas and Galphai. Firing of spontaneous action potentials (AP), calcium signaling and pulsatile GnRH secretion were similar to those of GT1-7 neurons. These findings demonstrate that subcellular localization, agonist-induced redistribution, homologous oligomerization, and coupling to the multiple G proteins of the GnRH-R receptor modulate electrical activity, second messengers, and neurosecretion of the hypothalamic GnRH neurons. Roles of Tyrosine322 and Tyrosine324 in GnRH Receptor Function *The gonadotropin-releasing hormone (GnRH) receptor is an anomalous member of the GPCR superfamily with several unique structural features. These include (a) the absence of a cytoplasmic C-terminal tail;(b) the replacement of Tyr by Ser in the conserved DRY motif located at the junction of transmembrane domain (TMD) III and the 2i loop;(c) the interchange of conserved Asp and Asn residues in TMDs II and VII;and (d) a relatively long and highly basic 1i loop. The Asn-Pro-any amino acid-Tyr (NPXY) sequence, where X represents an aliphatic amino acid, mediates interactions with intracellular effector molecules. The NPXY motif is present in TM VII of the GnRH-R and is conserved among 7 TM receptors. *GnRH receptor binding properties and cAMP signaling were analyzed in GT1-7 cells expressing the endogenous GnRH-R, and in COS-7 cells transfected with the mouse GnRH-R. Agonist stimulation of the wild type GnRH-R expressed in COS-7 cells using a pGFP2-N vector caused a monotonic dose-dependent increase in cAMP production. Deletion of Tyr322, or its change to Ser, increased GnRH binding capacity of the mutant receptor by 3-5 fold). In such receptors, GnRH-induced cAMP production was abolished, indicating the absence of Gs activation. GnRH-induced phosphorylation of mitogen-activated protein kinases (MAPKs) was also abolished in the muttant receptor, consistent with impairment of the protein kinase C (PKC)-dependent signaling pathway. GnRH-R binding activity was abolished when Tyr322 was substituted with Phe. Substitution of Tyr324 of the mouse GnRH-R with non-aromatic hydroxyl group-containing Ser caused loss of agonist binding. In contrast, when Tyr324 was replaced with Phe the mutant receptor retained high binding capacity and cAMP signaling comparable to that of the wild type GnRH-R. These findings demonstrate the importance of both Tyr322 and Tyr324 of the mouse GnRH receptor as crucial residues that determine its agonist binding and signaling properties. *GnRH-Induced Modulation of After-hyperpolarizing Current in GnRH Neurons In many central nervous system neurons, action potentials (APs) are followed by prolonged afterhyperpolarization (AHP) of the membrane potential that controls excitability and firing pattern. Two classes of AHPs and the underlying Ca2+-activated currents can be distinguished by their time course and pharmacological properties. IAHP underlies part of the medium AHP (mAHP) following single or repetitive APs, is sensitive to the bee venom toxin apamin, and exhibits rapid rise and decay (time constants 100 ms). In addition, endogenous GnRH receptors expressed in native and immortalized GnRH neurons initiate diverse signaling pathways by coupling to multiple G proteins. Such coupling is time- and dose-dependent, and switches between Gq, Gs, and Gi/o, in GnRH-stimulated GT1-7 neurons. These findings suggest that an agonist concentration-dependent switch in coupling of the GnRH-R between specific G proteins modulates neuronal Ca2+ signaling via Gs-cAMP stimulatory and Gi-cAMP inhibitory mechanisms. Activation of Gi may also inhibit GnRH neuronal function and episodic secretion by regulating membrane ion currents, possibly through activation of G protein-regulated inwardly rectifying potassium channels (GIRKs). Such channels are important for maintaining the resting potential and excitability of neurons. Accordingly, it is necessary to clarify the functional activities of these channels associated with regulation of the GnRH neuron, and its spontaneous electrical activity and episodic neuropeptide secretion. *Action potentials and the associated Ca2+ influx are followed by slow after-hyperpolarizations (sAHPs) caused by a voltage-insensitive, Ca2+-dependent K+ current. Slow AHPs are common in mammalian neurons and are present in both peripheral and central nervous systems. The firing of individual and/or bursts of action potentials (APs) in spontaneously active GnRH neurons is followed by hyperpolarization that lasts from several milliseconds (ms) to several seconds (s). Such hyperpolarization is mediated by the activation of two families of Ca2+-activated K+ channels. Big conductance (BK) channels contribute to action potential repolarization, whereas small conductance (SK) channels underlie the after-hyperpolarization (AHP) and mediate firing frequency and spike-frequency adaptation. Both fast after-hyperpolarizations (fAHP) and slow after- hyperpolarizations (sAHP) did not occur during spontaneous firing of fast rhythmic APs.*Small calcium-activated potassium current was recorded as a tail current by a depolarization step from -60mV to 40 mV. Agonist activation of GnRH neurons caused a significant increase in sAHP that was partially sensitive to apamin. Treatment of GnRH neurons with 10 nM GnRH increased the fast tonic APs, with unchanged decay constants for fAHP and mAHP. In contrast, treatment of neurons with 1 microM GnRH abolished mAHP current, but did not affect the fAHP current. That was followed by subthreshold after-depolarization potential (ADP) and significant reduction of the AP firing frequency. These data indicate that AP- and GnRH-driven Ca2+influx in GnRH neurons determines the profile of after-hyperpolarization currents and firing frequency.
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