GPCR – G – protein complex’s structure was crystalized at 2011 for the first time (Rasmussen et al., 2011). Heterotrimeric G Proteins are trimeric proteins inside the cell which are consisting of α, β, and γ subunits.
G Proteins take their name from their ability to bind and hydrolyze guanosine triphosphate (GTP) to yield guanosine diphosphate (GDP). When bound to GTP, they are stimulating the pathway they are included, whereas hydrolyzing GTP to GDP results in deactivation of the pathway. Upon activation of a GPCR, G-protein is activated by replacing GDP with GTP and G-protein α (Gα) subunit dissociates from G-protein βγ (Gβγ) subunits to start a signaling cascade via various secondary messengers, whereas Gβγ complex’s major role was thought to downregulate Gα’s activity by decreasing its affinity to GTP via changing Gα’s conformational shape, it is found that they are activating other pathways by interacting ion channels, kinases, and phospholipases. A demonstration of GPCR and G-protein interaction is shown at Figure 1
Some of the secondary messengers and effectors involved in signal transduction of G proteins involve phospholipase C (PLC), diacylglycerol (DAG), inositol triphosphate (IP3), and cyclic adenosine monophosphate (cAMP).
In the ligand-GPCR-G-protein bound state complex, it is found that G-protein has higher affinity for GTP than GDP and GPCR has multiple times more affinity to its ligand, pointing out that the structural changes in the complex units are ongoing upon complex formation. These changes include extracellular loops for they bind to ligands, intracellular ends for they interact with G proteins of GPCRs, and G-protein’s sites for receptor binding and GDP binding.
Gαparts of G proteins are classified into four main groups, namely Gαs, Gαi, Gαq, and Gα12. In short; Gαs is stimulatory, Gαi is inhibitory, Gαq is interacting with PLC, and Gα12 is involved in Rho family GTPase signaling (Figure 2).
These groups are also divided into subtypes depending on the ir alignments and effector types, in which main signal cascades are started or deactivated by, upon response to a ligand binding to a GPCR outside the cell, and resulting in cell’s biological responses. Table 1 represents known G-protein alpha parts.
Inhibitory G Proteins and Subtypes Go1 and Gi3
Inhibitory G protein subtype (Gi) alters cAMP levels inside the cell negatively by inhibiting adenylyl cyclic activity through interacting with io n channels rather than adenylyl cyclases. Gi subfamily includes G proteins Gi1, Gi2, Gi3, Go1, Go2, and Gz. This family of proteins is highly expressed; therefore, their activation is resulting in high levels of Gβγ complexes released from their Gα, which means inhibitory G-protein family also plays a triggering role in the Gβγ dependent signaling pathways.
Gi family is mostly found in nervous system and also affected by Pertussis toxin, which leaves inhibitory G proteins incapable of binding to their receptors (Wettschureck & Offermanns, 2005; Alberts et al., 2002). Also, since cAMP levels are related to insulin release, this family is also related to obesity and diabetes.
Coded by GNAO1 gene, alpha part of the trimeric Go1 protein is located on chromosome 16q12.2 and expressed abundantly in brain tissue. Mutations in its gene are shown to cause oncogenic features and epileptic encephalopathy by affecting the structure of the protein.
GNAI3 gene is coding for alpha part of inhibitory Gi3 protein and is located on 17q22-24. GNAI3 is shown to affect cytokinesis, proliferation, migration, invasion and apoptosis. Variants of G-protein subtype I3 (Gi3), namely c.118G>C and c.141C>A, are found to be related to auriculocondylar syndrome (ACS).
Methods for Detection of Interactions of GPCRs and G Proteins
Understanding of how this large family of GPCRs signal outside and inside the cell and interact with its ligands and counterparts is a quite important step to unravel mechanisms behind to improve drug development systems, correct targeting when medication is introduced, and beyond all, to understand a key signaling mechanism in many organisms to gain a strong insight about life. Within the scope of this aim, several experimental approaches have been developed to understand structures of these proteins. To decipher GPCR structure, construction of prediction models via sequence analysis showed that extracellular loops of these proteins are the least conserved regions whereas intracellular loops are highly conserved with the discovery of lengths of these loops varies suggesting an importance over interaction mechanisms of these receptors inside the cell.
Crystal structure of a GPCR was discovered for the first time in 2000, GPCR and its ligand at 2007 and with a G-protein complex at 2011. After these discoveries more dynamic studies to determine the interactions were established since the behavior of these proteins under cellular conditions and discovery of interactions with other possible proteins remained unknown.
Mass spectrometry is one of the methods used to characterize how G proteins are activated upon GPCR coupling. Using the data gathered from X-ray crystallography studies, nucleotide exchange mechanism of Gs upon coupling is provided via mass-spectrometry, particularly hydrogen-deuterium exchange mass spectrometry (HDXMS). While using HDXMS reveals more dynamic information about GPCR-G-protein interaction, studies in live cells are required to fully understand the characteristics.
Site-directed mutagenesis and coimmunoprecipitation (CoIP) studies are also widely used to determine interactions of GPCRs with G proteins. While these approaches are significantly helping over determining structural areas of interaction and the unknown proteins in the interaction complexes, they are still insufficient to shed a light on the mechanisms of interactions in live cells.
In vitro studies has limitations of being unnatural, proteins developed and investigated outside the cell do not behave exactly as in their natural environment. Another limitation is that the complexity of the behavior of the GPCRs and G proteins, it is known that a particular GPCR can act through more than one G-protein subtype and each coupling changes the outcome rapidly and significantly (Giulietti et al., 2014). Therefore, live cell studies are preferred over in vitro studies to fully understand the mechanisms of signaling of these proteins.
The most commonly used methods to track interactions of GPCRs and G proteins in live cells are Förster Resonance Energy Transfer (FRET) and Bioluminescent Resonance Energy Transfer (BRET). The most outstanding advantage of these methods is that they are applicable to live cell systems without interfering with the functionality. Second, energy transfer depends on the distance between molecules and this distance must be dramatically low for transfer to occur, this requirement brings enormous accuracy for tracking interactions between molecules. FRET relies on energy transfer between two fluorophores, whereas BRET uses an enzymatic donor. It is also possible to follow cAMP levels in live cells by using FRET and/or BRET methods via tracking conformational c hanges in the strategically placed biosensors (Denis et al., 2012). FRET’s advantage over BRET is that it allows tracking the events happening inside the cell, whereas, it is important to carefully pick the fluorophores used in FRET, for their spectra should be eligible to each other and for photobleaching, background noise due to autofluorescence of fluorophores may affect the studies.