28.2: At the cell membrane- receptors and receptor enzymes

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Learning Goals 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

G Protein-Coupled Receptors (GPCRs): Structure, Mechanism, and Diversity

• Describe the structural features of GPCRs (seven transmembrane α-helices, extracellular ligand-binding cavity, cytoplasmic G protein-interacting domain) and explain how agonist binding drives the conformational change that triggers GDP→GTP exchange in the Gα subunit and dissociation of Gα-GTP from Gβγ.
• Distinguish between stimulatory (Gsα) and inhibitory (Gi/oα) Gα subunits, and explain how the intrinsic GTPase activity of Gα provides a built-in timing mechanism that terminates the signal; describe how cholera toxin subverts this mechanism by ADP-ribosylating Arg in Gα.
• Compare the downstream effectors coupled to different Gα families (Gs→adenylyl cyclase→cAMP; Gq→phospholipase Cβ→DAG + IP3; G12/13→small GTPases), and explain why the existence of more than 20 Gα-like proteins, 800 GPCRs, and 13 PLC isoforms generates enormous combinatorial signaling diversity.
• Using cannabinoid receptors (CB1 and CB2) as a case study, explain how agonists, partial agonists, inverse agonists, antagonists, and negative allosteric modulators produce distinct receptor outputs, and describe how the THC:CBD ratio modulates psychotropic and therapeutic effects.

Receptor Tyrosine Kinases (RTKs): Structure, Activation, and Downstream Recruitment

• Explain how ligand binding to RTKs drives receptor dimerization, activation of intracellular kinase domains, and autophosphorylation, and contrast this mechanism with the indirect enzyme activation strategy used by GPCRs.
• Describe how autophosphorylated RTKs recruit SH2 domain-containing proteins—including kinases, phosphatases, transcription factors, and scaffolding proteins—to propagate and diversify the intracellular signal.

Signal Amplification, Allosteric Regulation, and Pharmacological Relevance

• Explain how enzymatic amplification at each step of a signaling cascade (a single activated enzyme producing many second-messenger molecules, each activating additional enzymes) magnifies the initial receptor-binding event.
• Explain how the MWC allosteric model—with pre-existing T (low-affinity) and R (high-affinity) conformational states—applies to GPCRs and other signaling proteins, and why more complex kinetic models are often required for ligand-gated ion channels and GPCRs in practice.

Introduction

We will now provide a more detailed description of cell signaling, beginning at the cell membrane and progressing inward toward the intracellular organelles, ultimately reaching the nucleus. The signal mediates changes in gene expression. Of course, this end is somewhat arbitrary, as the signal could propagate from the nucleus back to the membrane, where newly synthesized membrane proteins might be inserted or even exported, as in the case of secreted antibodies. It is difficult enough to keep track of all the molecular players, let alone determine their initial location in the cell and their final location if they undergo translocation. Signaling can also be more daunting for those with a focus on chemistry, who find the details of cellular structure and trafficking a bit overwhelming. Figure \(\PageIndex{1}\) shows a truncated view of the cell membrane, membrane proteins, and some of the organelles that we will visit throughout this chapter. To reiterate past learning, we will repeat the figure or its variants several times in this section.

Figure \(\PageIndex{1}\): Location of signaling events. Reference: Nucleus - https://commons.wikimedia.org/wiki/File:0318_Nucleus.jpg; Mitochondria: https://upload.wikimedia.org/Wikipedia/commons/f/f2/Blausen_0644_Mitochondria.png; Golgi: https://smart.servier.com/smart_image/golgi-apparatus-3/. Creative Commons Attribution 3.0 Unported License; Proteins, lysosome: https://smart.servier.com/. Creative Commons Attribution 3.0 Unported LicenseNucleus - https://commons.wikimedia.org/wiki/File:0318_Nucleus.jpg; Mitochondria: https://upload.wikimedia.org/Wikipedia/commons/f/f2/Blausen_0644_Mitochondria.png; Golgi: https://smart.servier.com/smart_image/golgi-apparatus-3/. Creative Commons Attribution 3.0 Unported License; Proteins, lysosome: https://smart.servier.com/. Creative Commons Attribution 3.0 Unported License

Receptors at the Cell Membrane

Let's start at the location where signaling almost invariably begins, in the cell membrane, where signals (hormones, neurotransmitters, nutrients, other cells) bind to cell surface transmembrane proteins shown in the red box in Figure \(\PageIndex{2}\).

Figure \(\PageIndex{2}\): Signaling events at the cell membrane

We have already discussed integral and peripheral membrane proteins in Chapter 11. When a ligand binds, the signaling event mediated by a transmembrane receptor might involve a change in the membrane protein's conformation, which propagates to its intracellular domain. Alternatively, the receptor might change its conformation to become a ligand-gated kinase (or possibly a phosphatase), a ligand-gated channel, or a pore. We will discuss signal-gated ion channels in Chapter 28.9, "Neural Signaling."

Ultimately, intracellular enzymes are activated within cells in response to external molecular signals. This provides amplification of the initial signal since a single activated enzyme undergoes multiple rounds of catalysis before it becomes inactivated. If multiple products (such as second messengers or phosphorylated proteins) are formed and these products activate multiple additional enzymes, the signal is further amplified.

Receptors with no kinase or transport activity - G Protein-Coupled Receptors (GPCRs)

One major type of signaling receptor is the G-protein-coupled receptor (GPCR). Over 800 GPCRs are encoded in the human genome, representing approximately 4-5% of the total number of protein-coding genes. The proteins below belong to five major families: rhodopsin, secretin, glutamate, adhesion, and frizzled/taste2. They don’t express enzymatic activity, but on binding, they can activate enzymes inside the cell by interacting through their cytoplasmic domains with G proteins (GTP-binding proteins) in the cytoplasm. GPCRs are called serpentine receptors because their single polypeptide chains have seven transmembrane-spanning α-helices. All GPCRs have similar yet slightly different structures, enabling them to interact with specific ligands. Many GPCRs bind unknown ligands and are therefore called orphan receptors. We will explore a few GPCRs in more detail below.

GPCRs that modulate the membrane enzyme adenylyl cyclase:

β-adrenergic receptor

The β-adrenergic receptor is a prototypical G-protein-coupled receptor (GPCR). Found in muscle, liver, and fat cells, it binds epinephrine and adrenaline, leading to energy mobilization and muscle activation, commonly referred to as the fight-or-flight response. The mechanism of GPCR activation is illustrated using the beta-adrenergic receptor as an example. The unoccupied adrenergic receptor is associated with a heterotrimeric G protein composed of α, β, and γ subunits. GDP is usually bound to the α subunit. Figure \(\PageIndex{3}\) shows a cartoon of the GPCR interacting with the heterotrimeric G protein.

Figure \(\PageIndex{3}\): GPCR-Heterotrimeric complex in the bilayer

When the hormone binds to the receptor, a conformational change propagates to the GPCR's cytoplasmic domain, altering its interaction with G_αβγ^. This causes a conformational change in the G_α subunit, leading to the exchange of bound GDP for GTP, which promotes the dissociation of the Gα-GTP complex from G_βγ. G_α-GTP then binds to and activates an adjacent membrane enzyme called adenylate cyclase, which produces a second messenger by converting ATP to cyclic AMP (cAMP). Figure \(\PageIndex{4}\) shows steps in the generic GPCR activation cycle.

Figure \(\PageIndex{4}\): Activation cycle of G-proteins by G-protein-coupled receptors. https://commons.wikimedia.org/wiki/F...PCR-Zyklus.png Creative Commons Attribution-Share Alike 3.0 Unported

The primary message binds to the GPCR (1 leading to state 2. Conformational changes in the cytoplasmic domain facilitate GTP exchange on the G_α subunit as state 3 transitions to state 4. In state 5 the G_α-GTP complex dissociates from G_βγ, which remains in the membrane. The G_α-GTP subunit is held and localized to the membrane (not evident in the above figure) through a lipid anchor attached through a post-translational modification.

Remember, the GPCR has no ligand-gated enzymatic activity. Yet it indirectly activates a membrane enzyme, adenylate cyclase, when the dissociated G_α-GTP binds to the cyclase. As long as GTP remains bound to the G_α subunit, it will continue to modulate the activity of adenylate cyclase. A built-in regulatory mechanism exists in the protein, as the G_α subunit possesses GTPase activity. The GTP will eventually be hydrolyzed, and the GDP-G_α subunit will lose affinity for its bound partner (adenylate cyclase) and return to the heterotrimeric G protein associated with the unbound receptor. GPCRs bind the signaling ligand (primary message) in a binding cavity localized at the extracellular face and between four of the transmembrane helices.

The activity and structure of GPCRs have been studied using natural ligands, such as hormones and neurotransmitters, as well as agonists, partial agonists, inverse agonists, and antagonists. As discussed previously, agonists bind to the natural ligand binding site and elicit a full or partial response (partial agonists). Inverse agonists bind and lower the response of a constitutively active receptor, and antagonists bind and prevent the normal response of an agonist. Approximately 35% of pharmaceutical drugs target GPCRs, but only about 15% of the ∼800 human GPCRs. The orphan GPCRs are increasingly targets for drug development. Most hormones and neurotransmitters work through GPCRs. Additionally, our primary senses of vision, olfaction (smell), and gustation (taste) function through GPCRs.

Figure \(\PageIndex{5}\) shows the structure of the beta 2-adrenergic:Gs complex with bound agonist. No membrane is shown.

Figure \(\PageIndex{5}\): Structure of the beta-adrenergic:Gs complex with bound agonist.

The GPCR is shown in cyan. The seven transmembrane helices should be obvious. The ligand is bound between them. The G_α subunit is shown in magenta, the G_β in dark blue, and the G_γ in orange/brown. The gray subunit is a Camelid antibody VHH fragment, a single-chain nanoantibody used to stabilize the conformation for crystallization.

The biggest conformational change that occurs when the GPCR binds an agonist is an outward movement (14 Å) at the intracellular domain of transmembrane segment 6 (TM6) and an extension of the TM5 helix. This leads to movement of the Gα's alpha-helical domain, enabling the exchange of GTP for GDP. Of course, multiple reactions determine the fraction of the G_α in the active GTP-bound state. These would include the relative cellular concentrations of free cytoplasmic GDP and GTP, their K_D values for the G_α, the rate constant for the hydrolysis of bound GTP, and the rate constants for the GDP ↔ GTP exchange.

Figure \(\PageIndex{6}\) shows an interactive iCn3D model of the Crystal structure of the β2 adrenergic receptor-Gs protein complex 3SN6

Figure \(\PageIndex{6}\): β-2 adrenergic receptor-Gs protein complex 3SN6 (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...8R4zKZCfKZy248

The GPCR is shown in green, the G_α in magenta, Gβ in blue, and the Gγ in brown. An agonist (spacefill) is shown in CPK colors near the outer leaflet.

Some bacterial toxins inactivate the GTPase activity of the Gα subunit, keeping it in the "stuck" state. For example, cholera toxin, an enzyme released by Vibrio cholerae, catalyzes the ADP ribosylation of an Arg in the G_α subunit by transferring everything but the nicotinamide from NAD⁺ to the Arg residue.

Since the G_α subunit stimulated the activity of adenylyl cyclases, it is often named a stimulatory G_α protein or G_sα. Figure \(\PageIndex{7}\) shows an interactive iCn3D model of the adenylyl cyclase activator G_sα with GTP-γ-S (1azt)

Figure \(\PageIndex{7}\): adenylyl cyclase activator G_sα with GTP-γ-S (1azt) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...T7MBqYnaxAH3SA

Now we can explore how the occupied GPCR, which again lacks enzymatic activity, activates adenylyl cyclase. Figure \(\PageIndex{8}\) shows a cartoon of the G_sα subunit bound to adenylyl cyclase as the G_βγ heterodimer remains associated with the membrane

Figure \(\PageIndex{8}\):

Adenylate cyclase converts ATP into the second messenger cyclic AMP (cAMP) as shown in Figure \(\PageIndex{9}\). The figure also shows how cAMP is broken down into AMP by the enzyme cAMP-specific 3',5'-cyclic phosphodiesterase (PDE). The latter step is necessary to control the lifetime of the second messenger cAMP.

Figure \(\PageIndex{9}\): Formation of cAMP by adenylyl cyclase and its degradation by phosphodiesterase

Why cAMP?

You might ask why cAMP, rather than just AMP, is nature's choice for GPCR signaling. AMP is a critical metabolic species. High concentrations of it signal an energy-depleted state. AMP is used in another signaling process to mobilize a response that adjusts the energy state of a cell, via the protein AMP-Activated Protein Kinase, which we will describe later. Many enzymes are also allosterically regulated by AMP.

Figure \(\PageIndex{10}\) shows an interactive iCn3D model of the membrane adenylyl cyclase bound to an activated stimulatory G_sα protein 6R3Q determined by cryo-EM.

Figure \(\PageIndex{10}\): membrane adenylyl cyclase bound to an activated stimulatory G protein 6R3Q (Copyright; author via source). Click the image for a popup or use this external link:https://structure.ncbi.nlm.nih.gov/i...foXY38deJSMDC6

The carboxyl-terminal cytoplasmic domain has the catalytic and allosteric sites.

Cannabinoid Receptors

In contrast to the beta-adrenergic receptor, which mediates activation of adenylate cyclase through G_sα, some G_α subunits inhibit adenylate cyclase when bound. These G_α subunits are called G_i/oα in contrast to the stimulatory subunits, G_sα. Additionally, G_α subunits interact with numerous proteins, not just adenylate cyclase. Examples include cannabinoid receptors.

Cannabinoid receptors are named after the exogenous and psychoactive drug Δ⁹-tetrahydrocannabinol (THC) that binds to the receptor. THC is the major phytocannabinoid (from plants) found in the Cannabis sativa plant and the marijuana derived from it. The other main cannabinoid in the plant is cannabidiol (CBD). Phytocannabinoids bind to two types of human cannabinoid (CB) receptors, CB1 and CB2. They have 44% amino acid and 68% homology in the entire protein and the transmembrane domain, respectively.

The phytocannabinoids exert their effects by binding to the human CB1 and CB2 receptors, whose endogenous ligands are two fatty acid derivatives: anandamide (AEA) and 2-arachidonoyl glycerol (2-AG). The structure of THC, CBD, and the two major endogenous ligands are shown in Figure \(\PageIndex{11}\).

Figure \(\PageIndex{11}\): Structure of agonist and antagonist for cannabinoid receptors

As cannabinoids and endocannabinoids have analgesic and other neurobiochemical properties, it might be possible to modulate their pathways to maximize pain relief offered by opioids but without their addictive effects.  Evidence in mice suggests that it is possible.  Levels of 2-AG (2-arachidonyl glycerol) can be increased in mice in vivo in the presence of a drug, JZL184, that inhibits monoacylglycerol lipase (MAGL), the enzyme that degrades 2-AG.  Then, when given morphine, the mice show decreased addictive behaviors but still experience the pain relief offered by morphine. These effects are mediated by the CB1 receptor for the endocannabinoid, as knocking out the gene decreases the effects of the MAGL inhibitor.  Figure \(\PageIndex{11a}\) below shows abbreviated pathways for the synthesis and degradation of 2-AG and AEA.  Another inhibitor, PF-3845, which similarly reduces AEA levels, did not have the same effect.

Figure \(\PageIndex{11a}\): abbreviated pathways for the synthesis and degradation of 2-AG and AEA.  Arlene Martínez-Rivera et al. Elevating levels of the endocannabinoid 2-arachidonoylglycerol blunts opioid reward but not analgesia. Sci. Adv.10,eadq4779(2024).DOI:10.1126/sciadv.adq4779. Creative Commons Attribution License 4.0 (CC BY)  https://creativecommons.org/licenses/by/4.0/

The cannabinoids from Cannabis sativa have a monoterpene isoprenyl group (C10) and a pentyl side chain (C5). The ligands are largely hydrophobic and are likely to access their binding sites on the receptor primarily through lateral movement in the membrane. The receptors differ most in the N-terminal extracellular loop, which is also involved in ligand binding.

Figure \(\PageIndex{12}\) shows an interactive iCn3D model of the class A GPCR Cannabinoid Receptor-Gi Complex Structure with bound agonist (6KPF)

Figure \(\PageIndex{12}\): Class A GPCR Cannabinoid Receptor-GiComplex Structures (6KPF) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...B16mYTKPMhXmg8

The gray protein is a nanobody used to stabilize the protein during crystallization. The agonist is AM12033, which is similar to AM11542 in the figure above, but with a -C=N terminus instead of a bromide.

THC and CBD

Cannabis sativa contains the psychoactive drug, Δ⁹-tetrahydrocannabinol (THC), which is a partial agonist for CB1 (binds with reported K_i values of 10 or 53 nm)and CB2 (K_i = 40 nm). Its psychoactive effects on mental activity, as well as pain and appetite, are well known. In contrast, cannabidiol (CBD) is the main, non-psychoactive cannabinoid. It has a significantly lower affinity for the recombinant CB1 (K_i = 1.5 µM) and CB2 (Ki = 3.7 µM). It appears to be a partial CB1 antagonist and a weak inverse agonist at CB2. It has also been shown that CBD is a negative allosteric modulator of the agonistic effects of THC and 2AG. The actual psychotropic effects of combining THC and CBD are complicated and not well understood.

Figure \(\PageIndex{13}\) shows an interactive iCn3D model of human CB1 in complex with agonist AM11542 (5XRA)

Figure \(\PageIndex{13}\): Human CB1 in complex with agonist AM11542 (5XRA). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...wDv9fXNPaMhwm9

The complex shows a significant conformational change compared to the receptor bound to an antagonist. This includes a 50% reduction in the volume of the binding pocket and an increase in the receptor's surface area that binds to G proteins.

How much of a receptor is bound with a cannabinoid depends on the concentration of the cannabinoid ligand and the K_i for the drug. The amount of THC and CBD depends on the genetics of the plant, which has been engineered to greatly decrease THC production (in the hemp plant used for nonpharmacological commercial properties) or increase either THC or CBD production at the expense of the other.

The synthesis of THC and CBD proceeds through a common precursor, CBGA (cannabigerol acid). Two key flavoproteins, Δ⁹-tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) convert this common precursor CBGA into two new precursors, Δ⁹-THCA and CBDA, respectively. This final synthetic step involves an oxidative cyclization reaction with O₂, producing H₂O₂. Spontaneous, non-catalyzed decarboxylation and rearrangements of Δ⁹-THCA and CBDA lead to the final products, THC and CBD. This last process occurs upon exposure to heat, which happens during smoking and baking, and at a slower rate during storage.

Commercially used preparations of THC and CBD for medicinal purposes vary widely in concentrations. THC concentration ranges for pain management (<5-10%) are much lower than those for psychotropic effects (<15%), with values of 21% or higher often found in "recreational" cannabis. High-potency THC strains can contain up to 25-30% THC by dry weight. For strains modified for CBD production, the maximal amount is about 25%. Even though CBD appears to be a partial antagonist for CB1, it appears that the ratio of THC:CBD is important in modulating the "high" or intoxicating state of THC. A ratio of THC:CBD of just over 1:1 leads to synergism or enhancement of the acute effects of THC, whereas ratios of THC:CBD of 1:2 to 1:6 seem to have the least intoxicating effects. However, CBD decreases the psychotic symptoms of THC and also decreases the memory changes associated with THC. CBD is also an allosteric modulator of the μ-opioid receptor.

At present, there are no structures of CBD bound to its cannabinoid receptors. Figure \(\PageIndex{14}\) shows an interactive iCn3D model of the CBD-bound full-length rat transient receptor potential vanilloid 2 (TRPV2) in nanodiscs (6U88)

Figure \(\PageIndex{14}\): CBD-bound full-length rat TRPV2 in nanodiscs (6U88). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...BJcXBriq49LtQA

This receptor is a calcium-permeable, non-selective cation channel that is activated at high temperatures (50 °C) but not by vanilloids like capsaicin. Hence, it acts as a "noxious high temperature" receptor.

 

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Now let's make it even more complicated. There are more than 20 different G_α-like proteins, classified into four major families.

• G_s and G_i regulate adenylyl cyclase
• G_q activates phospholipase Cβ (described below). There are four members given these strange names: G_q, G₁₁, G₁₄, and G_15/16
• G_12/13 activate small GTPase protein (described in Chapter 28.5)

The G_α protein involved in light sensation is named transducin, while those involved in odorant detection and taste are called G_olfactory and G_gustatory, respectively.

As we add more variants of each signaling component, the origin of signaling system complexity becomes evident. For example, the neurotransmitter serotonin binds to its receptor, a G-protein-coupled receptor (GPCR). Instead of gating the protein open to ion flow (as with other ligand-gated ion channels in the activation of neurons, as we will see in Chapter 28.9), it interacts with two different alpha subunits, G_s, which leads to activation of adenylyl cyclase, and G₁₂, which interacts with other small GTP-binding proteins called GEFs (we will also see these later).

GPCRs modulate the activity of the membrane enzyme phospholipase C.

These receptors utilize the same mechanism to activate the membrane enzyme adenylyl cyclase. When the primary signal is bound to the GPCR, a conformational change is propagated to the cytoplasmic domain of the GPCR, altering its interaction with G_αβγ in which the alpha subunit is a member of the G_α(q) family. This causes a conformational change in the G_α subunit, leading to the exchange of bound GDP with GTP, and subsequently promoting the dissociation of the Gα-GTP complex. G_α-GTP then binds to and activates an adjacent membrane enzyme, phospholipase C (PLC), which cleaves membrane phospholipids to produce two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (IP₃). Their structures are shown in Figure \(\PageIndex{15}\).

Figure \(\PageIndex{15}\):Activation of phospholipase C

There appear to be 13 types of mammalian phospholipase Cs, divided into six isotypes: β, γ, δ, ε, ζ, and η. Phospholipase C is also named 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase.

Figure \(\PageIndex{16}\) shows an interactive iCn3D model of the G_α(q)-phospholipase C-β 3 structure (4GNK). The magenta structure is the G_α(q) protein with bound GDP in spacefill. The cyan structure is the Pleckstrin Homology (PH) domain of the protein. This domain targets proteins to inositol phospholipids in the membrane, but it does not appear to have this function in this protein.

Figure \(\PageIndex{16}\): G_α(q)-phospholipase C-β-3 structure (4GNK) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?5kiejFu8KgzXsiP9A

Note that in contrast to adenylyl cyclase, PLC is a peripheral, not an integral membrane protein. It is found in the cytoplasm as well as associated with the inner leaflet of the cell membrane, where its main activities, regulating and cleaving PIP₂, occur. PLC localizes to lipid rafts enriched in PIP₂. Figure \(\PageIndex{17}\) shows the domain structure of phospholipase C β3

Figure \(\PageIndex{17}\) Domain structure of phospholipase C β3

The N-terminal PH_14 represents the Pleckstrin Homology (PH) domain, which is among the top 15 of all domains in the human genome. All PLCs except PLCζ have this domain. Note that in another example of complexity, PLC-β binding to the inner leaflet does not require PIP2. The iCn3D model above indeed shows that binding to the membrane appears to depend on adjacent structures, rather than the PH domain (cyan) specifically.

Table \(\PageIndex{1}\) below shows characteristics of common signals that signal through GPCRs.

signal	vasopressin	epinephrine	light	odorant	odorant	sweet tastant
receptor	VR	β-adrenergic	rhodopsin	odorant receptor 1	odorant receptor 2	sweet receptor
Ga-like subunit	Gi	Gs	transducin	Golfactory	Golfactory	Ggustatory
coupled enzyme	adenylate cyclase	adenylate cyclase	phosphodiesterase	phospholipase C	adenylate cyclase	adenylate cyclase
2nd messenger	decrease cAMP	increase cAMP	decrease cGMP	increase IP3	increase cAMP	increase cAMP
protein affected	decrease PrK-A	increase PrK-A	dec. Ca, Na perm.	inc. Ca perm	inc.Ca, Na perm	dec. K perm

Table \(\PageIndex{1}\): Characteristics of common signals that work through GPCRs.

Changeux and Edelstein reviewed the MHC model 40 years after its conception and supported its application to signal transduction processes. They include, as signaling molecules, not only hemoglobin, but also regulatory enzymes (aspartate transcarbamylase, phosphofructokinase, LDH, glycogen phosphorylase), membrane receptors (acetylcholine receptor, rhodopsin), and nuclear receptors (lac repressor, steroid hormone receptors). In all these signaling proteins, residues distant from the "active" site participate in binding to allosteric ligands. Often, the allosteric site is on a separate domain that can be cleaved from the protein and still retain allosteric ligand-binding properties. The proteins also consist of multiple subunits easily related by distinct symmetry axes.

Allosteric ligands often bind in cavities in subunit interfaces along symmetry axes. In general, crystal structure analyses show that low-affinity T and high-affinity R forms of the signaling proteins exist.  They are accompanied by minor tertiary structure changes in individual subunits (i.e., perfect symmetry in all subunits is not preserved on the binding of an allosteric ligand). In neurotransmitter membrane receptors, these two states can correspond to open and closed states (for ion flux), and open conformations of these proteins are often found in mutant forms. However, for many ligand-gated ion channels and G-protein-coupled receptors (serpentine), kinetic analyses reveal more complex forms than can be represented by a simple two-state (R and T) model. High-resolution microscopy shows evidence for nonsymmetrical quaternary structural changes. These changes can be observed in the absence of a ligand, supporting the MWC concept that allosteric ligands select specific conformational states, leading to equilibrium shifts in the unliganded receptor toward a higher-affinity state. More refined methods of structural analysis will presumably show more evidence of subtle tertiary changes in the proteins that are preludes to quaternary structural changes. Yet the simplicity of the MWC model for explaining many features of signaling proteins remains.

Soluble GPCRs designed from scratch

We discussed ways to design new proteins from scratch in Chapter 4.14.  One example is the design of soluble GPCRs that retain the protein's structure and functional features.  Soluble versions of GPCRs may enable easier and more effective testing of drug candidates. Figure \(\PageIndex{x}\) shows an interactive iCn3D model of the de novo designed soluble GPCR-like fold GLF_32 (8OYY).

Figure \(\PageIndex{x}\): de novo designed soluble GPCR-like fold GLF_32 (8OYY).  (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...KScWHJaXBA84PA

The structure is coded by hydrophobicity, with green indicating the most hydrophobic.  A transparent surface is also shown.  The green hydrophobic side chains are mostly buried and form nonpolar contacts with adjacent packed helices.  The surface is mostly nonpolar.  Compare this to the surface of the GPCR used as a model for this structure, the Metabotropic Glutamate Receptor 5 (mGlu5, 6FFI).

 

Receptors with signal-gated kinase activity - Receptor Tyrosine Kinases (RTK)

Why bother binding a primary message to a GPCR and going through multiple steps before activating a membrane enzyme like adenylate cyclase? Wouldn't it be more efficient to have the membrane receptor as a ligand-activated enzyme? Such is the case with special membrane receptors called Receptor Tyrosine Kinases (RTKs). There are about 90 tyrosine kinases in the human genome, of which 58 are RTKs. Figure \(\PageIndex{18}\) shows the family domain structure of the RTKs.

Figure \(\PageIndex{18}\): Family domain structure of the RTKs. Yamaoka, T.; Kusumoto, S.; Ando, K.; Ohba, M.; Ohmori, T. Receptor Tyrosine Kinase-Targeted Cancer Therapy. Int. J. Mol. Sci. 2018, 19, 3491. https://doi.org/10.3390/ijms19113491. Creative Commons Attribution License

Note that the insulin receptor (InsR) is a dimer of two monomeric insulin receptor chains. Figure \(\PageIndex{19}\) provides a more detailed view of the domain structure of the epidermal growth factor receptor (EGFR).

Figure \(\PageIndex{19}\): Domain structure of the EGFR.

The domain (red/brown) immediately after the blue domain is the transmembrane domain. Furin is a cellular endoprotease. The green represents L domains, which comprise the ligand-binding site. Each L domain consists of a single-stranded right-hand beta-helix.

Here is the cascade of events for signaling through EGFR: The transmembrane has ligand-dependent tyrosine kinase activity. Binding of the hormone EGF causes receptor dimerization, bringing the intracellular kinase domains (yellow PK_Try_Ser_Thr) together and activating them. When active, they can autophosphorylate (phosphorylate themselves) or other proteins. When the receptor is autophosphorylated, other proteins can bind to the receptor's cytoplasmic domain, where they are phosphorylated. The target substrates phosphorylated by receptor tyrosine kinases are proteins that share a common 100-amino-acid domain called SH (src homology), based on structural homology with another cytoplasmic protein, Src. Src is an intracellular Tyr kinase activated when it binds through 2 SH domains to an autophosphorylated receptor Tyr kinase. Specifically, the SH2 domain has been shown to bind tyrosine-phosphorylated peptides. These domains target proteins to the autophosphorylated receptor Tyr kinase. Many proteins involved in signal transduction have SH2 domains. Some of these proteins also have catalytic domains with kinase activity. Others have phosphatase, transcription factor, or scaffolding domains.

Figure \(\PageIndex{20}\) shows the hormone-dependent dimerization of RTKs, their autophosphorylation, and the recruitment of proteins with SH2 domains.

Figure \(\PageIndex{20}\): Activation of receptor tyrosine kinases (RTKs)

It is easier to envision how a GPCR is activated by binding its target hormone than for RTKs. GPCRs are single-chain proteins that pass through the membrane using 7-transmembrane helices. RTKs have a single transmembrane helix. The dimeric form of the RTK exhibits additional flexibility in the short region between the extracellular and transmembrane domains, enabling the conformational changes required to activate the intracellular kinase domains.

The crystal structure of the full EGFR is not known, given the difficulties in crystallizing membrane proteins that span the membrane with a single alpha helix. However, separate structures of the dimeric extracellular domain and the intracellular kinase domains are known.

Figure \(\PageIndex{21}\) shows an interactive iCn3D model of the dimeric extracellular and transmembrane domains of the epidermal growth factor receptor (3NJP).

Figure \(\PageIndex{21}\): Dimeric extracellular and transmembrane domains of the epidermal growth factor receptor (3NJP) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...iw1V4T8rsfxDZ7

The two EGFRs are shown in blue and magenta. The two bound EGFs are shown in cyan.

Figure \(\PageIndex{22}\) shows an interactive iCn3D model of a dimer of the intracellular dimeric EGFR kinase domains in complex with an ATP analog-peptide conjugate (2GS6)

Figure \(\PageIndex{22}\): Dimeric intracellular EGFR kinase domains in complex with an ATP analog-peptide conjugate (2GS6) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...iw1V4T8rsfxDZ7

The two EGFR kinase domains are shown in cyan and magenta. The ATP analogs in each domain (spacefill) are thiophosphoric acid O-((adenosyl-phospho)phospho)-S-acetamidyldiester. The peptide substrates (green sticks) are 13-mer peptides with a tyrosine (sticks, labeled Y, minus the hydroxyl groups) connected to the ATP analog.

We have introduced the essential players of the cell membrane involved in signal transduction.

• GPCRs, which are not enzymes but which activate the bound heterotrimer G protein Gα subunit, which then can activate or inhibit the integral membrane protein adenylate cyclase or activate the membrane-bound enzyme protein kinase C. These enzymes produce second messengers cAMP (adenylate cyclase), diacylglycerol (DAG), and IP₃ (phospholipase C)
• Receptor tyrosine kinases, ligand-activated receptor kinases, can, on ligand-induced dimerization, autophosphorylate themselves or other target proteins in the cell.

In the next chapter section, we will explore the next downstream effects in signaling, mediated by the second messengers cAMP, DAG, and IP3, and the substrates phosphorylated by the ligand-activated receptor tyrosine kinases.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

Cell surface signaling is initiated when extracellular ligands—hormones, neurotransmitters, nutrients, or sensory stimuli—bind to transmembrane receptor proteins without themselves entering the cell. Two major classes of receptors dominate this chapter. G protein-coupled receptors (GPCRs), the largest family of signaling receptors with over 800 members in the human genome, are seven-pass transmembrane proteins that lack intrinsic enzymatic activity but activate heterotrimeric G proteins at their cytoplasmic face. Agonist binding drives a conformational change that opens a nucleotide exchange site on the Gα subunit, causing GDP to be replaced by GTP; the activated Gα-GTP then dissociates from Gβγ and modulates downstream effectors. The built-in GTPase activity of Gα ensures signal termination, a mechanism exploited destructively by cholera toxin, which permanently locks Gα in the active state through ADP-ribosylation.

The identity of the Gα subunit determines downstream outcomes. Gsα stimulates adenylyl cyclase to produce cAMP, while Gi/oα inhibits it; Gqα activates phospholipase Cβ, which cleaves the membrane lipid PIP2 into two distinct second messengers—diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The cannabinoid receptors CB1 and CB2 illustrate how the same receptor family can be engaged by endogenous ligands (anandamide, 2-AG), plant-derived phytocannabinoids (THC, CBD), and synthetic compounds, each with a strikingly different pharmacological profile depending on whether they act as agonists, partial agonists, inverse agonists, or negative allosteric modulators.

Receptor tyrosine kinases (RTKs), the second major receptor class, combine ligand binding and enzymatic activity in a single protein. Ligand binding triggers receptor dimerization, juxtaposing the intracellular kinase domains and enabling reciprocal autophosphorylation. Phosphotyrosine residues then serve as docking sites for SH2-domain-containing proteins, including kinases, phosphatases, transcription factors, and scaffolding proteins, thereby rapidly expanding the signaling network. A key advantage of both GPCR and RTK systems is enzymatic amplification: a single activated receptor can generate many second-messenger molecules or phosphorylate many substrate proteins, each of which can further amplify the signal downstream. Together, these membrane receptor systems form the molecular gateway through which environmental information is converted into coordinated intracellular responses.