Any such amplifying cascade of stimulatory signals requires counterbalancing mechanisms at every step of the cascade to restore the system to its resting state when stimulation ceases. As emphasized earlier, the response to stimulation can be rapid only if the inactivating mechanisms are also rapid. Cells therefore have efficient mechanisms for rapidly degrading (and resynthesizing) cyclic nucleotides and for buffering and removing cytosolic Ca2+, as well as for inactivating the responding enzymes and ion channels once they have been activated. This is not only essential for turning a response off, but is also important for defining the resting state from which a response begins.
Each protein in the signaling relay chain can be a separate target for regulation, including the receptor itself, as we discuss next.
As discussed earlier, when target cells are exposed to a high concentration of a stimulating ligand for a prolonged period, they can become desensitized, or adapted, in several different ways. An important class of adaptation mechanisms depends on alteration of the quantity or condition of the receptor molecules themselves.
For GPCRs, there are three general modes of adaptation (see Figure 15–20): (1) In receptor sequestration, they are temporarily moved to the interior of the cell (internalized) so that they no longer have access to their ligand. (2) In receptor down-regulation, they are destroyed in lysosomes after internalization. (3) In receptor inactivation, they become altered so that they can no longer interact with G proteins.
In each case, the desensitization of the GPCRs depends on their phosphorylation by PKA, PKC, or a member of the family of GPCR kinases (GRKs), which includes the rhodopsin-specific kinase RK involved in rod photoreceptor desensitization discussed earlier. The GRKs phosphorylate multiple serines and threonines on a GPCR, but they do so only after ligand binding has activated the receptor, because it is the activated receptor that allosterically activates the GRK.
500 G-protein (transducin) molecules are activated 500 cyclic GMP phosphodiesterase molecules are activated 105 cyclic GMP molecules are hydrolyzed 106–107 Na+ ions per second are prevented from entering the cell for a period of ~1 second membrane potential is altered by 1 mV
Figure 15–41 Amplification in the light-induced catalytic cascade in vertebrate rods. The red arrows indicate the steps where amplification occurs, with the thickness of the arrow roughly indicating the magnitude of the amplification.
Figure 15–42 The roles of GPCR kinases (GRKs) and arrestins in GPCR desensitization.
A GRK phosphorylates only activated receptors because it is the activated GPCR that activates the GRK. The binding of an arrestin to the phosphorylated receptor prevents the receptor from binding to its G protein and also directs its endocytosis (not shown). Mice that are deficient in one form of arrestin fail to desensitize in response to morphine, for example, attesting to the importance of arrestins for desensitization.
As with rhodopsin, once a receptor has been phosphorylated by a GRK, it binds with high affinity to a member of the arrestin family of proteins (Figure 15–42).
The bound arrestin can contribute to the desensitization process in at least two ways. First, it prevents the activated receptor from interacting with G proteins. Second, it serves as an adaptor protein to help couple the receptor to the clathrin-dependent endocytosis machinery (discussed in Chapter 13), inducing receptor-mediated endocytosis. The fate of the internalized GPCR–arrestin complex depends on other proteins in the complex. In some cases, the receptor is dephosphorylated and recycled back to the plasma membrane for reuse. In others, it is ubiquitylated, endocytosed, and degraded in lysosomes (discussed later).