G-AlphaS Signaling
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G-AlphaS Signaling
G-Proteins are heterotrimers, consisting of Alpha, Beta and Gamma subunits, and are involved in signaling to distinct effectors. Heterotrimeric G-Proteins convey extracellular signals that activate 7-transmembrane-spanning GPCRs (G-Protein-Coupled Receptors) to the inside of cells, communicating this information to effector proteins and thus initiating changes in cell behaviour. GPCRs constitute a large and diverse family of proteins whose primary function is to transduce extracellular stimuli into intracellular signals. GPCRs recognize a variety of ligands and stimuli including Peptide and non-peptide Hormones and Neurotransmitters, Chemokines, Prostanoids and Proteinases, Biogenic amines, Nucleosides, Lipids, Growth factors, Odorant molecules and Light. GPCRs turn on G-Proteins by promoting the binding of the activating nucleotide GTP in exchange for GDP on the G-Alpha subunit. In the inactive heterotrimeric state, GDP is bound to the G-Alpha subunit. Upon activation, GDP is released, GTP binds to G-Alpha, and subsequently G-Alpha-GTP dissociates from G-BetaGamma and from the receptor. Both G-Alpha-GTP and G-BetaGamma are then free to activate downstream effectors. The duration of the signal is determined by the intrinsic GTP hydrolysis rate of the G-Alpha-subunit and the subsequent reassociation of G-Alpha-GDP with G-BetaGamma (Ref.1 & 2).

Four classes of Heterotrimeric G-Alpha proteins, G-AlphaI/O, G-AlphaS, G-AlphaQ/11, and G-Alpha12/13, couple heptahelical GPCRs to effectors to relay extracellular signals into eukaryotic cells. G-AlphaS, like all G-Alpha subunits, consist of two domains: a GTPase domain that includes the sites for guanine nucleotide binding and receptor and effector interaction and a helical domain that may be important to maintain guanine nucleotide binding. Long and short forms of G-AlphaS result from alternative splicing of exon 3. G-AlphaS family of G-Proteins consists of 3 members: GNAS (G-AlphaS), GNASXL (G-AlphaS-XL), GNAL (G-AlphaS-Olf). The most well characterized function of G-AlphaS is as a signal transducer between GPCR and generation of cAMP (Cyclic Adenosine 3,5-monophosphate) by AC (Adenylyl Cyclase). Upon activation, G-AlphaS subunit get separated from GN-Beta (Guanine Nucleotide-Binding Protein-Beta) and GN-Gamma (Guanine Nucleotide-Binding Protein-Gamma) subunits and exhibit distinctive regulatory features on the nine tmACs in order to regulate intracellular cAMP levels. Once active, the tmACs (transmembrane Adenylyl Cyclase) produces the second messenger cAMP in response to a wide range of signal transduction pathways. Three main targets of cAMP are PKA (Protein Kinase-A), the GTP-exchange protein, EPACs (Exchange Protein Activated by cAMP) and the CNG (Cyclic-Nucleotide Gated Ion Channel). CNG activation by cAMP provides passage to Ca2+ influx. cAMP activate Rap1A (Ras-Related Protein-1A) and Rap1B (Ras-Related Protein Rap1B) through the PKA-independent and EPAC-dependent pathway. cAMP activates cAMP-GEFI (cAMP-Regulated Guanine Nucleotide Exchange Factor-I)/EPAC1 and cAMP-GEFII (cAMP-Regulated Guanine Nucleotide Exchange Factor-II)/EPAC2 that in turn activate Rap1A and Rap1B, respectively. Rap1A and Rap1B then forms an active complex with BRaf (v-Raf Murine Sarcoma Viral Oncogene Homolog-B1) for MEK1/2 (MAPK /ERK Kinase-1/2) activation finally resulting in Elk1 activation via ERK (Extracellular Signal-Regulated Kinase). Rap1 also inhibits c-Raf. Rap1A and Rap1B further stimulate Rap1 and Rap2 pathways that are vital for cell survival. Apart from CNG, PKA, and EPACs, other direct targets of cAMP includes, PDE (Phosphodiesterase), mTOR (Mammalian Target of Rapamycin), p70S6K/RPS6KB1 (Ribosomal Protein-S6 kinase-70kDa-Polypeptide-1), PLA2 (Phospholipase-A2). Although cAMP directly regulates the activities of many molecules, PKA appears to be the major read-out for cAMP and is the predominant cellular effector of cAMP (Ref.3, 4 & 5).

In its inactivated state, PKA exists as a tetrameric complex of two Catalytic subunits (PKAC) and a Regulatory (PKAR) subunit dimer. PKA is generally anchored to specific cellular locations within the cell by specific proteins called AKAPs (A Kinase-Anchoring Proteins). When both binding sites on the R subunits are occupied by cAMP (Cyclic Adenosine 3,5-monophosphate), the R subunits undergo a conformational change, which lowers their affinity for the C-subunits. This results in the dissociation of the holoenzyme complex and renders the enzyme active. The catalytic subunits are then free to phosphorylate specific target proteins. Thus, PKA is activated by cAMP to transfer phosphates from ATP (Adenosine Triphosphate) to protein substrates. PKA can phosphorylate several substrates. Some of the major substrates of PKA include L-Type CaCn (Calcium Channels), RyR (Ryanodine Receptor), GSK3 (Glycogen Synthase Kinase-3) and APC (Anaphase-Promoting Complex). RyR1 on the SR (Sarcoplasmic reticulum) is the major Ca2+ (calcium) release channel required for skeletal muscle EC (Excitation-Contraction) coupling. RyR1 function is modulated by proteins that bind to its large cytoplasmic scaffold domain, including PKA. PKA phosphorylation of RyR1 at Ser2843 activates the channel by releasing FKBP12. RyR1, PKA hyperphosphorylation correlated with impaired SR Ca2+ release. Serine 21 in GSK3Alpha (Glycogen Synthase Kinase-3-Alpha) and Serine 9 in GSK3Beta (Glycogen Synthase Kinase-3-Beta) are also physiological substrates of PKA. PKA physically associates with, phosphorylates, and inactivates both isoforms of GSK3, thus prevents Oncogenesis and neurodegeneration.PKA also inactivates APC. Inactivation of APC by PKA helps to maintain control cell proliferation and anaphase initiation and late mitotic events, respectively, thereby checking the degradation cell cycle regulators. PKA also phosphorylates transcription factor CREB (cAMP Responsive Element Binding Protein) at Ser133, which in turn allows recruitment of the coactivator CBP (CREB-Binding Protein), which interacts with transcription initiation factor IIB in the basal transcription complex. PKA also inhibit Adducin and take part in cytoskeletal regulation. Thus, PKA is important for an increasing number of physiological processes such as cAMP regulation of ion channels in the nervous system, regulation of the cell cycle which involves microtubule dynamics, chromatin condensation and decondensation, nuclear envelope disassembly and reassembly, Steroidogenesis, reproductive function, immune responses and numerous intracellular transport mechanisms (Ref.6, 7 & 8).

Besides activating AC , G-AlphaS also stimulate the kinase activity of downregulated c-Src and HCK, members of Src-family tyrosine kinases. G-AlphaS bind to the catalytic domain and change the conformation of Src, leading to increased accessibility of the active site to substrates. Src activated by direct interaction with GPCRs or components of the GPCR signaling machinery, including G-AlphaS subunit is most clearly associated with the regulation of G-protein function, receptor desensitization, and endocytosis. Src-dependent phosphorylation of heterotrimeric G-proteins may alter their GTPase activity and affect the association of G-Alpha and G-Beta Gamma subunits. G-AlphaS may also activate Calcium channels and take part in calcium signaling. The lifespan of the G-AlphaS subunit can be markedly reduced by RGS (Regulator of G protein signalling) proteins.  RGS proteins are multi-functional, GTPase-accelerating proteins that promote G-Alpha subunit GTP hydrolysis, thereby directly terminating G-Alpha subunit signalling and indirectly terminating G-BetaGamma dimer signalling (through Alpha subunit binding).  Direct binding of RGS2 to G-AlphaS is at least partly responsible for the inhibitory effect of RGS2 on cAMP accumulation. Alternatively, there is evidence for a direct interaction between RGS2 and AC. Even after 40 years of study there are new details emerging for the G-AlphaS pathway. G-Protein-mediated signal transduction is a complex, highly organized signaling network with diverging and converging transduction steps at the ligand-receptor, receptor-G-Protein, and G-Protein-effector interfaces. Unraveling the complexities of these cascades will help in the design of experiments that eventually may reveal new mechanisms of disease states, targets for drugs, and sites and mechanisms of action of anesthetics on these important signaling pathways (Ref.1, 9 & 10).