Approximately 800 of the roughly 23,000 genes thought to comprise the human genome code for G protein-coupled receptors. Although many of these are odorant receptors in olfactory neurons (see Chapter 32), many others are receptors for well-characterized neurotransmitters used throughout the nervous system. Despite their enormous diversity, all G protein-coupled receptors consist of a single polypeptide with seven characteristic membrane-spanning regions (serpentine receptors) (Figure 11–4).
G protein-coupled receptors contain seven membrane-spanning domains.
The β2-adrenergic receptor shown here is representative of G protein-coupled receptors, including the β1-adrenergic and muscarinic acetylcholine (ACh) receptors and rhodopsin. It consists of a single subunit with an extracellular amino terminus, intracellular carboxy terminus, and seven membrane-spanning α-helixes. The binding site for the neurotransmitter lies in a cleft in the receptor formed by the transmembrane helixes. The amino acid residue aspartic acid (Asp)-113 participates in binding. The part of the receptor indicated in brown associates with G protein α-subunits. Two serine (Ser) residues in the intracellular carboxy-terminal tail are sites for phosphorylation by specific receptor kinases, which helps inactivate the receptor. (Adapted, with permission, from Frielle et al. 1989.)
The number of substances that act as second messengers in synaptic transmission is much fewer than the number of transmitters. Approximately 100 substances serve as transmitters; each can activate several types of receptors on the cell surface. The few second messengers that have been well characterized fall into two categories, intracellular and transcellular. Intra cellular messengers are molecules whose actions are confined to the cell in which they are produced. Trans cellular messengers are molecules that can readily cross the cell membrane and thus can leave the cell in which they are produced to act as intercellular signals, or first messengers, on neighboring cells.
A Family of G Proteins Activates Distinct Second-Messenger Pathways
The first G protein, Gs (where "s" stands for stimulatory), was identified more than 30 years ago by Martin Rodbell, Al Gilman, and their colleagues. Since that time a large family of G proteins has been identified. The G proteins are associated with the inner leaflet of the plasma membrane, where they interact with G protein-coupled receptors.
The G proteins that couple receptor activation to intracellular effectors are trimers that consist of three subunits: α, β, and γ (Figure 11–2). The α-subunit is only loosely associated with the membrane and is usually the agent that couples the receptor to its primary effector enzyme. The β- and γ-subunits form a strongly bound complex that is more tightly associated with the membrane. As we shall learn later in this chapter, the βγ complex of G proteins can also regulate the activity of certain ion channels directly.
Approximately 20 types of α-subunits have been identified, 5 types of β-subunits, and 12 types of γ-subunits. G proteins with different α-subunits couple different classes of receptors and effectors, and therefore have different physiological actions. The β-adrenergic receptor activates adenylyl cyclase by acting on Gs proteins; these contain the αs type of α-subunit. Some muscarinic ACh receptors inhibit the cyclase by acting on Gi proteins (where "i" stands for inhibitory); these contain the αi type subunit. Still other G proteins (Gq/11 proteins, which contain αq- or α11-subunits) activate phospholipase C and probably other signal transduction mechanisms not yet identified. The Go protein, which contains the αo-subunit, is expressed at particularly high levels in the brain. Compared with other organs of the body, the brain contains an exceptionally large variety of G proteins. Even so, because of the limited number of classes of G proteins, one type of G protein can often be activated by different classes of receptors.
The known effector targets for G proteins are more limited than the types of G proteins. Important effectors include certain ion channels that are activated by the βγ complex, adenylyl cyclase in the cAMP pathway, phospholipase C in the diacylglycerol-inositol polyphosphate pathway, and phospholipase A2 in the arachidonic acid pathway. Each of these effectors (except for the ion channels) initiates changes in specific target proteins within the cell, either by generating second messengers that bind to the target protein or by activating a protein kinase that phosphorylates it. Despite their differences, second-messenger pathways activated by G protein signaling share a common design (Figure 11–5).
Synaptic second-messenger systems involving G protein coupling follow a common sequence.
The signal transduction pathways illustrated here involve similar steps (left). Chemical transmitters arriving at receptor molecules in the plasma membrane activate a closely related family of G proteins (the transducers) that activate different enzymes or channels (the primary effectors). The activated enzymes produce a second messenger that activates a secondary effector or acts directly on a target (or regulatory) protein.
cAMP system. This pathway can be activated by a transmitter-bound β-adrenergic receptor, which acts through the Gs protein αs-subunit to activate adenylyl cyclase. Adenylyl cyclase produces the second messenger cAMP, which activates PKA. The G protein here is termed Gs because it stimulates the cyclase. Some receptors activate a Gi protein that inhibits the cyclase.
Phosphoinositol system. This pathway, activated by a type 1 muscarinic acetylcholine (ACh) receptor, uses the Gq or G11 type of G protein (with αq- or α11-subunits, respectively) to activate a primary effector, phospholipase Cβ (PLCβ) . This enzyme hydrolyzes the phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2) , yielding a pair of second messengers: diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) . In turn, IP3 releases Ca2+ from internal stores, whereas DAG activates protein kinase C (PKC). The drop in membrane PIP2 levels can directly alter the activity of some ion channels.
Direct G protein-gating. This pathway represents the simplest synaptic mechanism for G protein-coupled receptor action. Acetylcholine (ACh) acting on type 2 muscarinic receptors activates the Gi protein, leading to functional dissociation of the αi-subunit and βγ complex. The βγ complex interacts directly with a G protein-gated inward-rectifying K+ channel (GIRK), leading to channel opening and membrane hyperpolarization.
Hydrolysis of Phospholipids by Phospholipase C Produces Two Important Second Messengers, IP3 and Diacylglycerol
Many important second messengers are generated through the hydrolysis of phospholipids in the inner leaflet of the plasma membrane. This hydrolysis is catalyzed by three enzymes—phospholipase C, phospholipase D, and phospholipase A2 —named for the ester bonds they hydrolyze in the phospholipid. The phospholipases each can be activated by different G proteins coupled to different receptors.
The most commonly hydrolyzed phospholipid is phosphatidylinositol 4,5-bisphosphate (PIP2) , which typically contains the fatty acid stearate esterified to the glycerol backbone in the first position and the unsaturated fatty acid arachidonate in the second:
Activation of receptors coupled to Gq or G11 stimulates phospholipase C, which leads to the hydrolysis of PIP2 (specifically the phosphodiester bond that links the glycerol backbone to the polar head group) and production of two second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) . DAG, which is hydrophobic, remains in the membrane when formed, where it recruits the cytoplasmic protein kinase C (PKC). PKC and DAG together with certain membrane phospholipids form an active complex that can phosphorylate many protein substrates in the cell, both membrane-associated and cytoplasmic (Figure 11–6A). Activation of some isoforms of PKC requires elevated levels of cytoplasmic Ca2+ in addition to DAG (Box 11–1).
Hydrolysis of phospholipids in the cell membrane activates three major second-messenger cascades.
A. The binding of transmitter to a receptor activates a G protein that activates phospholipase Cβ (PLCβ) . This enzyme cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 is water-soluble and diffuses into the cytoplasm, where it binds to a receptor-channel on the smooth endoplasmic reticulum, the IP3 receptor, to release Ca2+ from internal stores. DAG remains in the membrane, where it activates protein kinase C (PKC). Membrane phospholipid is also a necessary cofactor for PKC activation. Some isoforms of PKC also require Ca2+ for activation. PKC is composed of a single protein molecule that has both a regulatory domain that binds DAG and a catalytic domain that phosphorylates proteins on serine or threonine residues.
B. The Ca2+/calmodulin-dependent protein kinase is activated when Ca2+ binds to calmodulin. The Ca2+/calmodulin complex then binds to a regulatory domain of the kinase, causing its activation. The kinase is composed of many similar subunits (only one of which is shown here), each having both regulatory and catalytic functions. The catalytic domain phosphorylates proteins on serine or threonine residues. (ATP, adenosine triphosphate; C, catalytic subunit; COOH, carboxy terminus; H2N, amino terminus; R, regulatory subunit.)
Box 11–1 Isoforms of Protein Kinase C
At least nine isoforms of protein kinase C (PKC) have been found in nervous tissue. Rather than having regulatory and catalytic functions in different subunits, like PKA, most PKC isoforms contain regulatory and catalytic domains in a single continuous polypeptide chain (see Figure 11–6A).
Two functionally interesting differences have thus far been found among these isoforms. The so-called major forms (α, βI, βII, and γ) all have a calcium-binding site and are activated by Ca2+ ions together with diacylglycerol. The minor forms (eg, δ, ε, and ζ) lack the calcium-binding domain, and therefore their activity is independent of Ca2+.
The second interesting difference is that, of the major forms, only PKCγ is activated by low concentrations of arachidonic acid, a membrane fatty acid, although all the isoforms respond to diacylglycerol or phorbol esters (plant toxins that bind to PKC and promote tumors).
With one exception, PKC isoforms also contain a site between the regulatory and catalytic domains that is sensitive to proteolysis. High levels of cytoplasmic Ca2+ can activate proteases that cleave PKC at this site, releasing a cytoplasmic form of PKC called protein kinase M (PKM). This protein fragment is constitutively active because it lacks the regulatory domain. As a result, elevations in Ca2+ can lead to prolonged activation of the kinase.
Long-lasting activation of PKC can also be produced through expression of the gene encoding PKC. This isoform is unique in that it lacks a regulatory domain and is therefore constitutively active. Expression of PKC produces persistent PKC activity in hippocampal neurons during the induction of long-term potentiation, which is thought to underlie certain forms of learning and memory in the hippocampus (see Chapter 67).
The second product of the phospholipase C pathway, IP3, stimulates the release of Ca2+ from intracellular membrane stores in the lumen of the smooth endoplasmic reticulum. The membrane of the reticulum contains a large integral membrane macromolecule, the IP3 receptor, which forms both a receptor for IP3 on its cytoplasmic surface and a Ca2+-permeant channel that spans the membrane of the reticulum. When this macromolecule binds IP3 the channel opens, releasing Ca2+ into the cytoplasm (Figure 11–6A).
The increase in intracellular Ca2+ triggers many biochemical reactions and opens calcium-gated channels in the plasma membrane. Calcium can also act as a second messenger to trigger the release of additional Ca2+ from internal stores by binding to another integral protein in the membrane of the smooth endoplasmic reticulum, the ryanodine receptor (so called because it binds the plant alkaloid ryanodine, which inhibits the receptor; in contrast, caffeine opens the ryanodine receptor). Like the IP3 receptor to which it is distantly related, the ryanodine receptor forms a Ca2+-permeant channel that spans the reticulum membrane; however, cytoplasmic Ca2+, not IP3, gates the ryanodine receptor-channel.
Calcium often acts by binding to the small cytoplasmic protein calmodulin. An important function of the calcium/calmodulin complex is to activate the Ca2+/calmodulin-dependent protein kinase (CaM kinase). This enzyme is a complex of many similar subunits, each containing both regulatory and catalytic domains within the same polypeptide chain. When the Ca2+/ calmodulin complex is absent, the C-terminal regulatory domain of the kinase binds and inactivates the catalytic portion. Binding to the Ca2+/calmodulin complex causes conformational changes of the kinase molecule that unfetter the catalytic domain for action (Figure 11–6B). Once activated, CaM kinase can phosphorylate itself through intramolecular reactions at many sites in the molecule. Autophosphorylation has an important functional effect: It converts the enzyme into a form that is independent of Ca2+/calmodulin and therefore persistently active even in the absence of Ca2+.
Persistent activation of protein kinases is a general and important mechanism for maintaining biochemical processes underlying long-term changes in synaptic function associated with certain forms of memory. In addition to the persistent activation of Ca2+/calmodulin-dependent protein kinase, PKA can also become persistently active following a transient increase in cAMP, because of the enzymatic degradation of its regulatory subunits through the ubiquitin pathway. The decline in regulatory subunit concentration results in the long-lasting presence of free catalytic subunits, even after cAMP levels have declined, leading to the continued phosphorylation of substrate proteins. PKC can also become persistently active through proteolytic cleavage of its regulatory and catalytic domains or expression of a PKC isoform that lacks a regulatory domain.
Hydrolysis of Phospholipids by Phospholipase A2 Liberates Arachidonic Acid to Produce Other Second Messengers
Phospholipase A2 hydrolyzes phospholipids distinct from PIP2 by cleaving the fatty acyl bond between the 2′ position of the glycerol backbone and arachidonic acid. This releases arachidonic acid, which is then converted through enzymatic action to one of a family of active metabolites called eicosanoids, so called because of their 20 (Greek eicosa) carbon atoms.
Three types of enzymes metabolize arachidonic acid: (1) cyclooxygenases, which produce prostaglandins and thromboxanes; (2) several lipoxygenases, which produce a variety of metabolites to be discussed below; and (3) the cytochrome P450 complex, which oxidizes arachidonic acid itself as well as cyclooxygenase and lipoxygenase metabolites (Figure 11–7).
Three phospholipases generate distinct second messengers by hydrolysis of phospholipids containing arachidonic acid.
Pathway 1. Stimulation of G protein-coupled receptors leads to activation of phospholipase A2 (PLA2) by the free βγ subunit complex. Phospholipase A2 hydrolyzes phosphatidylinositol (PI) in the plasma membrane, leading to the release of arachidonic acid, a 20-carbon fatty acid that is a component of many phospholipids. Once released, arachidonic acid is metabolized through several pathways, three of which are shown. The 12- and 5-lipoxygenase pathways both produce several active metabolites; the cyclooxygenase pathway produces prostaglandins and thromboxanes. Cyclooxygenase is inhibited by indomethacin, aspirin, and other nonsteroidal anti-inflammatory drugs. Arachidonic acid and many of its metabolites modulate the activity of certain ion channels.
Pathway 2. Other G proteins activate phospholipase C (PLC), which hydrolyzes PI in the membrane to generate DAG (see Figure 11–6). Hydrolysis of DAG by a second enzyme, diacyl glycerol lipase (DAGL), leads to production of 2-arachidonyl glycerol (2-AG), an endocannabinoid that is released from neuronal membranes and then activates G protein-coupled endocannabinoid receptors in the plasma membrane of other neighboring neurons.
Pathway 3. Elevation of intracellular Ca2+ activates phospholipase D (PLD), which hydrolyzes phospholipids that have an unusual polar head group containing arachidonic acid (N-arachidonylphosphatidylethanolamine [N-arachidonyl PE]). This action generates a second endocannabinoid termed anandamide (arachidonylethanolamide). (HPETE, hydroperoxyeicosatetraenoic acid.)
Arachidonic acid and its metabolites are soluble in lipids and thus readily diffuse through membranes. Therefore, in addition to acting within the cell in which they are produced, these substances can act on neighboring cells, including a presynaptic neuron. In this way they act as transcellular synaptic messengers (discussed in the next section).
Synthesis of prostaglandins and thromboxanes in the brain is dramatically increased by nonspecific stimulation such as electroconvulsive shock, trauma, or acute cerebral ischemia (localized absence of blood). Many of the actions of prostaglandins are mediated in the plasma membrane by a family of G protein-coupled receptors. The members of this receptor family can activate or inhibit adenylyl cyclase or activate phospholipase C.
Lipoxygenases introduce an oxygen molecule into the arachidonic acid molecule, generating hydroperoxyeicosatetraenoic acids (HPETEs). These metabolites are synthesized in response to depolarization of brain slices with high concentrations of extracellular K+, glutamate, or NMDA. The compounds 5-HPETE, 12-HPETE, and some of their downstream metabolites modulate certain ion channels. These metabolites may also be important in mediating pain sensation by activating transient receptor potential (TRP) ion channels in certain sensory neurons (see Chapter 5). They also can act as transcellular second messengers, a function that appears to be important for long-term synaptic changes in the hippocampus.