G Protein Receptors

The body uses 7 membrane-spanning serpentine receptors for an astounding variety of biological signalling functions. Receptors on the cells lining our tongue convey taste. Hundreds of distinct receptor species in the cells of our olfactory bulbs in our nose convey information about the presence of odors (odorant ligands). A carotenoid molecule related to vitamin A is bound in the ligand position of rhodopsin in the rods and cones of our eyes where it serves to pick up photons, alter its conformation, and cause the receptor to which it is bound to release signals into the rod/cone cytoplasm that result in our perception of light. These serpentine receptors are of very ancient lineage. Baker's yeast cells communicate their sexual identity to each other by release of polypeptide mating factors. The cell surface receptors that recognize these mating factors are once again 7 membrane-spanning serpentine receptors! We will explore another well-studied example of a ligand/serpentine receptor pair.

Epinephrine

This one involves the ligand epinephrine also known as adrenaline, which is released by the adrenal glands above the kidneys in response to very stressful stimuli. Once released, epinephrine courses throughout our blood stream and adsorbs to specific receptors on the surfaces of cells in various tissues throughout the body. The result is the establishment of the primitive mammalian fight / flight reaction. This reaction increases heart rate, decreases blood flow to gut, increases blood flow to skeletal muscles, and increases blood glucose by causing liver and muscle cells to break down glycogen and release resulting glucose into the circulation. How does epinephrine/adrenaline evoke all these responses? Acting as a ligand, it binds to its own receptor displayed on the surface of a variety of cell types throughout the body. This beta adrenergic receptor is a 7 membrane-spanning, serpentine receptor embedded in the plasma membranes of these cells. As is the case with the growth factor receptors described earlier, the epinephrine ligand is not internalized into the cell. Instead, while bound for a short period of time to its receptor, it causes the latter to release biochemical signals into the cell cytoplasm. Serpentine receptors like the beta adrenergic receptor do not depend upon receptor dimerization (as described above) in order to transduce signals across the plasma membrane. Instead, single receptor molecules will change their 3 dimensional steric configuration in response to ligand binding. This steric shift affects the configuration of the cytoplasmic domains of the receptor, that is the loops of receptor protein that protrude into the cytoplasm.

Cytoplasmic signal transduction

The beta adrenergic receptor communicates with the cytoplasm by stimulating a second protein, which is known as a G protein for reasons that will become clear. The G protein normally lies near the receptor in an inactive, quiet state. When the receptor gets activated by ligand binding, it will rapidly poke the G protein. The G protein responds by switching itself on into an active state. Once in the active state, the G protein will send signals further into the cell. However, the G protein will remain in the active state for only a brief period of time, after which it will shut itself off. In effect, the G protein acts like a binary switch, a light switch which,once turned on, will remain on for a limited period of time before it flips itself off.

The G protein's two states (ON or OFF) are determined by the guanine nucleotide that it binds (whence the term G protein). When it is inactive it binds GDP; when active, it binds GTP. Accordingly, the resting, OFF form of the G protein sits around with its bound GDP. When a ligand- activated receptor pokes it, the G protein releases its bound GDP and allows a GTP molecule to jump aboard. This GTP-bound form of the G protein represents the active ON configuration of the G protein. While in the ON state, it releases downstream signals. After a short period of time (seconds or less), the G protein will then hydrolyze its own GTP down to GDP, thereby shutting itself off. This hydrolysis represents a negative feedback mechanism which ensures that the G protein is only in the active, signal- emitting ON mode for a short period of time.

Signalling Cascades

We will make a brief excursion into the downstream signalling pathway (often called a signal cascade) that is triggered by the active G protein. In fact the G protein is formed from 3 distinct protein subunits, termed alpha, beta, and gamma. When in its inactive OFF state, 3 subunits are bound together; the a subunit has the job of binding the guanine nucleotide, in this case GDP. When the beta adrenergic receptor activates the G protein, the alpha subunit releases GDP, binds GTP and falls away from the beta and gamma subunits.

Once this happens, the GTP-bound a subunit also loses affinity for the receptor, dissociates from it, and moves over and pokes yet another nearby protein, the enzyme adenylate cyclase, which until this time has been inactive. Once it is poked by the active, GTP-binding oc subunit of the G protein, the adenylate cyclase enzyme gets activated and does its job: it cyclizes ATP into 3'5' cyclic AMP. This reaction involves the release of the beta and gamma phosphates from the ATP and the linking of the surviving a phosphate (still attached to the 5' hydroxyl of ribose) to the 3' hydroxyl as well, forming a circular or cyclic structure, whence the term ''cyclic adenosine monophosphate'' or simply cAMP.

After a several second encounter with the adenyl cyclase enzyme, the alpha subunit of the G protein will hydrolyze its bound GTP and release the adenyl cyclase, thereby reverting to an inactive OFF signalling state. It will then rejoin the beta and gamma subunits that it deserted earlier in the game. The adenyl cyclase, no longer being poked by the activated a subunit of the G protein, will shut down and stop making cAMP from ATP. The whole cycle has resulted in only a brief pulse of signalling, in this case the production of several hundred cAMP molecules made by the adenylate cyclase during its brief period of activity.

Once made, the cAMP molecules act as intracellular hormones.

Such intracellular hormones are sometimes termed second messengers, in that they can diffuse throughout the cytoplasm and carry information to distant sites. In the absence of signalling events like that described above, the levels of cAMP in the cell are very low. They are raised for a brief period of time after the cAMP is created as described above and spread out through the cell. (Eventually, as you might imagine, the released cAMP is broken down to AMP, shutting down the whole process.)

At various sites throughout the cell, the cAMP binds to yet another enzyme which is a serine/threonine protein kinase, termed the cAMP- dependent kinase or simply ''A kinase''. A kinase, once activated at various sites in the cell, now can phosphorylate target substrate proteins on specific serine/threonine residues, thereby activating these target proteins. In the case of cells in the liver which store large amounts of glycogen, the high cAMP concentrations enable A kinase to

  1. phosphorylate and thereby activate an enzyme that activates glycogen phosphorylase which in turn breaks down glycogen into glucose-l-phosphate molecules; and
  2. it phosphorylates glycogen synthase, and in this way turns it off, thereby preventing the reconversion of the released glucose to glycogen. These two changes together ensure the mobilization of glucose through the breakdown of glycogen stored in the liver. A number of other reactions are triggered as well that together contribute to the fight/flight response.

There is enormous signal amplification in this cascade. A single epinephrine molecule (present at 1O-10M) may cause the activation of dozens of alpha subunits of proteins. Each of these in turn will activate the synthesis of a single adenylate cyclase, and each of these in turn will synthesize hundreds of cAMP molecules. Each of these in turn can activate a cAMP-dependent kinase that will on its own right modify hundreds of target molecules in the cell.