As mentioned above, G-proteins may terminate their own activation due to their intrinsic GTP→GDP hydrolysis capability. However, this reaction proceeds at a slow rate (≈0.02 times/sec) and, thus, it would take around 50 seconds for any single G-protein to deactivate if other factors did not come into play. Indeed, there are around 30 isoforms of RGS proteins that, when bound to Gα through their GAP domain, accelerate the hydrolysis rate to ≈30 times/sec. This 1500-fold increase in rate allows for the cell to respond to external signals with high speed, as well as spatial resolution due to limited amount of second messenger that can be generated and limited distance a G-protein can diffuse in 0.03 seconds. For the most part, the RGS proteins are promiscuous in their ability to deactivate G-proteins, while which RGS is involved in a given signaling pathway seems more determined by the tissue and GPCR involved than anything else. In addition, RGS proteins have the additional function of increasing the rate of GTP-GDP exchange at GPCRs, (i.e., as a sort of co-GEF) further contributing to the time resolution of GPCR signaling.
In addition, the GPCR may be desensitized itself. This can occur as:
# a direct result of ligand occupation, wherein the change in conformation allows recruitment of GPCR-Regulating Kinases (GRKs), which go on to phosphorylate various serine/threonine residues of IL-3 and the C-terminal tail. Upon GRK phosphorylation, the GPCR's affinity for β-arrestin (β-arrestin-1/2 in most tissues) is increased, at which point β-arrestin may bind and act to both sterically hinder G-protein coupling as well as initiate the process of receptor internalization through clathrin-mediated endocytosis. Because only the liganded receptor is desensitized by this mechanism, it is called homologous desensitization
# the affinity for β-arrestin may be increased in a ligand occupation and GRK-independent manner through phosphorylation of different ser/thr sites (but also of IL-3 and the C-terminal tail) by PKC and PKA. These phosphorylations are often sufficient to impair G-protein coupling on their own as well.
# PKC/PKA may, instead, phosphorylate GRKs, which can also lead to GPCR phosphorylation and β-arrestin binding in an occupation-independent manner. These latter two mechanisms allow for desensitization of one GPCR due to the activities of others, or heterologous desensitization. GRKs may also have GAP domains and so may contribute to inactivation through non-kinase mechanisms as well. A combination of these mechanisms may also occur.
Once β-arrestin is bound to a GPCR, it undergoes a conformational change allowing it to serve as a scaffolding protein for an adaptor complex termed AP-2, which in turn recruits another protein called clathrin. If enough receptors in the local area recruit clathrin in this manner, they aggregate and the membrane buds inwardly as a result of interactions between the molecules of clathrin, in a process called opsonization. Once the pit has been pinched off the plasma membrane due to the actions of two other proteins called amphiphysin and dynamin, it is now an endocytic vesicle. At this point, the adapter molecules and clathrin have dissociated, and the receptor is either trafficked back to the plasma membrane or targeted to lysosomes for degradation.
At any point in this process, the β-arrestins may also recruit other proteins—such as the non-receptor tyrosine kinase (nRTK), c-SRC—which may activate ERK1/2, or other mitogen-activated protein kinase (MAPK) signaling through, for example, phosphorylation of the small GTPase, Ras, or recruit the proteins of the ERK cascade directly (i.e., Raf-1, MEK, ERK-1/2) at which point signaling is initiated due to their close proximity to one another. Another target of c-SRC are the dynamin molecules involved in endocytosis. Dynamins polymerize around the neck of an incoming vesicle, and their phosphorylation by c-SRC provides the energy necessary for the conformational change allowing the final "pinching off" from the membrane.