Файл: Extracellular and Intracellular Signaling (книга).pdf

11.2 Cellular Signaling

Life of an organism at the biochemical level can be thought of as a collection of biological events, some occurring sequentially in time and some in parallel. Each of the biological events can, in turn, be broken down into one or more signaling cascades that usually consist of multiple signaling processes separated in space and time. How a specific cell in an organism will behave depends critically on this spatio-temporal separation of signaling processes. Cellular signaling broadly refers to these highly evolved networks of signaling events and cascades that allow a cell to function.

Cellular signaling has been studied for more than 100 years now and our knowledge of its complexity at multiple levels has been greatly enhanced through advances in many di erent areas of biology.1–3 It still appears that we may have barely opened the ‘‘Pandora’s box’’ as the current knowledge seems unable to explain the beautiful richness of the complexity of life observed on land and especially in the oceans. One of the many great examples of signaling complexity manifested in nature is the dynamic camouflage ability of cuttlefish, where highly coupled signaling cascades enable these mollusks to replicate not only the color of their environment but also its visual pattern and texture (depth) onto their skin to blend in with that environment.4,5

11.2.1Types of Signaling

Any signaling network or cascade is a series of biochemical processes, where each process is initiated by the appearance of a signal which is followed by its sensing, processing and transmission as another signal or signals for the next downstream process in the signaling cascade. The signal may appear either inside or outside the cell for processing. Extracellular signals are usually sensed and processed by plasma membrane proteins. Intracellular signals are processed by soluble proteins or membrane proteins in the plasma membrane or those on the surface or cell organelles.

The spatio-temporal separation of signaling processes and cascades mentioned earlier allows one to classify signaling processes into the following types that depend on the spatial origin of the signal in an organism and its reach within the organism:

a)Endocrine signaling: In this long-range signaling, signal molecules such as hormones are released by a cell and travel long distances (via bloodstream in animals or vascular system in plants) to cause an e ect in a di erent part of the organism. Processing of sensory signals like light, taste and smell can also be considered endocrine.

b)Paracrine signaling: This is a short-range version of endocrine signaling, where the signal produced by a cell is sensed locally, e.g. neurotransmitters that are processed by proximal neurons.

c)Juxtacrine signaling: In this signaling process, the signal is membrane bound on one cell and is sensed by a receptor on the adjacent cell,

e.g. membrane proteins on a cell membrane can be sensed by a Notch protein on the neighboring cell.

d)Autocrine signaling: In this signaling process, cells release a signal molecule outside the cell, which is sensed by a membrane protein on the same cell leading to self-stimulation, e.g. breast cancer cells release transforming growth factor alpha (TGF-a) that interact with its epidermal growth factor (EGF) receptor.

e)Intracrine signaling: In this signaling process, the signal molecule is generated inside the cell and sensed by another receptor from inside the cell.

f)Electrical signaling: This specialized signaling process propagates an electrical potential along the length of the cell and occurs on a long spatial scale. The cells that use this process are the neurons of the animal nervous system, which are unusually long cells.

Any signaling cascade may be made up of one or more of the abovementioned signaling processes.

11.2.2Membrane Proteins in Signaling

The diversity of signals is immense. Chemical signals are molecules ranging greatly in size from the very small (like oxygen molecule, adrenaline, etc.), to peptides (like cytokines) and large proteins. Non-chemical signals include photons that are absorbed by cis-retinal-rhodopsin complex in the retina and initiate a cascade of processes that start in the cell and end in the brain with the perception of vision. Di erent proteins have evolved along with the signaling processes to sense this broad spectrum of signals.

Spatial separation of signaling cascades in an organism is achieved by cells expressing di erent receptors on their surface as well as inside the cell. Cell surface receptors (membrane proteins) enable signal transduction across the plasma membrane by converting an extracellular signal into one or more intracellular signaling cascades. Three main classes of membrane proteins dominate TM signal transduction:6

a)Ion-channel receptors (ICRs): These proteins are responsible for sensing neurotransmitter molecules or voltage gradients across the membrane, as upon binding to the signal molecules or sensing the membrane potential

these receptors undergo a conformational change that opens or closes a channel and allows specific ions to cross the plasma membrane.7

b)Enzyme-linked receptors (ELRs): These are a diverse class of single-pass TM proteins that contain an extracellular ligand binding site and an intracellular catalytic/enzyme-binding site with a guanylyl cyclase, phos-

phatase, serine/threonine kinase or tyrosine kinase activity. Receptor tyrosine kinases dominate this class.8

c)G protein-coupled receptors (GPCRs): These form the largest superfamily of membrane proteins that undergo ‘‘signal-specific’’ conformational changes upon activation by a diverse set of extracellular signals. These

conformational changes in the receptor are transmitted to cytoplasmic G proteins and b-arrestins for downstream signal transmission and potential diversification for a physiological response as will be discussed later.

GPCRs are integral membrane proteins with an extracellular N-terminus and seven TM helices connected by loop regions. They use their N-terminus, extracellular loops and extracellular facing TM portions to sense their signals. As a single protein family, they interact with the most diverse set of signals from sensory signals (vision, taste, smell, pheromones, etc.) to large signal molecules (other proteins).

GPCRs are the focus of this chapter because being embedded in the plasma membrane they sit at the top of complex signaling cascades as gatekeepers and use their conformational flexibility to amplify, diversify and select downstream signaling pathways inside the cell with amazing specificity. This role has also implicated them in almost all disease mechanisms9 and about 30% of the approved drugs use them as targets to block or activate a whole signaling pathways in cells.10

In the next section we will describe what is known about GPCRs, both experimental and computational e orts to determine their structures, biochemical studies probing their downstream signaling e ects, structural as well functional implications of their signal sensing (ligand binding) properties and challenges in drug design aimed at GPCR targets.

11.3 G Protein-Coupled Receptors

GPCRs are integral membrane proteins with seven TM helices connected by three extracellular loops (ECLs) and three intracellular loops (ICLs). They form the largest superfamily in the human genome with B800 GPCRs identified, including B370 non-sensory receptors.11 A variety of bioactive molecules, including biogenic amines, peptides, lipids, nucleotides, hormones and proteins modulate GPCR activity to e ect regulation of essential physiological processes (e.g. neurotransmission, cellular metabolism, secretion, cell growth, immune defense and di erentiation). Thus, many important cell recognition and communication processes involve GPCRs. Due to mediating numerous critical physiological functions, GPCRs are involved in all major disease areas including cardiovascular, metabolic, neurodegenerative, psychiatric, cancer and infectious diseases.9 GPCRs represent 30–50% of the current drug targets for activation (by agonist drugs) or inhibition (by antagonists or inverse agonists). It is estimated that the B80 GPCR-targeting drugs currently marketed account for B$50 billion annual sales. Many of these drugs have annual sales 4$2 billion. Target evaluation, lead identification and optimization of GPCR assays have accelerated progress in identifying multiple subtypes for many GPCRs with specific cell and tissue functions. A detailed structural understanding of their function (activation) will have a tremendous and broad impact in many areas.12

Box 11.1 Reprinted by permission from Macmillan Publishing Ltd.: George et al., Nat. Rev. Drug Discov., 1(10), 808–820. Copyright 2002.

homologous to each other unless they bind to the same ligands and, apart from the seven-TM helix topology, nothing appears to be common across all receptors.

Rhodopsin (Family 1) receptors share some common sequence motifs like D(E)RY at the bottom of TM3, WXPFF motif in TM6, NPXXY motif in TM7 and some conserved prolines usually in the middle of many TMs that produce kinks in their helices. Small molecule ligands typically bind in the extracellular facing half of the TM regions and peptides/proteins bind mainly to the extracellular loops and N-terminus. There is a highly conserved disulfide bridge between cysteines in ECL2 and top of TM3.

Secretin/Adhesion (Family 2) receptors have a long N-terminal ectodomain that binds to ligands and contains many conserved cysteines, which can help the long N-terminus to form a stable tertiary structure (see Box 11.1). These receptors don’t share any sequence motifs with Family 1 receptors even in the TM regions, so it is not obvious if they will have the same TM bundle topology of Family 1 receptors.

Glutamate (Family 3) receptors have a long N-terminus and a long C- terminus as well. Most receptors use their long N-terminus to bind to their

shows the structures of bovine rhodopsin, human b2 adrenergic, turkey b1 adrenergic and human adenosine A2A receptors.23 They all share the same TM topology (relative positions of TM helices), but can di er (sometimes significantly) in helix tilts and rotations as will be discussed in the next section.

An invertebrate GPCR (squid rhodopsin) has also been crystallized, which showed unusually long TM regions 5 and 6.29 At least two more human GPCR structures are expected this year (dopamine D3 and chemokine CXCR4 receptors) and significantly more in the next decade. Progress is also being made in developing solid-state NMR techniques30 for GPCR structure determination. This rapid growth in GPCR crystal structures since 2007 is beginning to provide insight into the structural biology of these proteins,23,31 however, the progress is expected to remain slow due to intrinsic flexibility of these versatile receptors, which prevents them from packing into ordered crystals.

11.3.1.2Structural Diversity of Current GPCR Structures

The seven-TM helix topology of GPCRs presents unique advantages and challenges for the quantification of sequence-structure relationships. Many comparative modeling programs can predict structures of globular proteins (with 30% or higher sequence identity to a crystallized protein) to a reasonable accuracy, as the belief is that a major fraction of structural folds is now known for globular proteins. The same cannot be said for membrane proteins in general. GPCRs, however, can be thought of as having one structural fold, consisting of seven TM helices interconnected by intracellular and extracellular loops. The TM helices display high sequence conservation as compared to the loop regions as expected (see Table 11.1).

The table shows the sequence identity (Table 11.1A) and sequence similarity (Table 11.1B) (similarity using BLOSUM62,32 where two residues are considered similar if the corresponding substitution element in the BLOSUM62 matrix is 4 0) for the five GPCR sequences that have been crystallized.

To quantify the relationship between sequence and structure for GPCRs, we need to characterize the known structures using some standard geometrical parameters. As crystal structures don’t provide absolute membrane orientation of GPCRs, we use their orientation as predicted by the OPM (Orientation of Proteins in Membrane) database,33 which aligns each newly deposited membrane protein structure to an implicit membrane maximizing the free energy of membrane insertion. The middle of the membrane corresponds to the z ¼ 0 plane or the hydrophobic plane. Each GPCR structure can then be characterized by the six orientation parameters of the seven helices relative to this plane. Figure 11.2A shows how the helix position and tilt are defined. Helix position (R) on the hydrophobic plane is then given by x and y. Value h corresponds to the hydrophobic center residue from the helix that will be