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Membrane & Signaling Processes
As they relate to Terms 1 and 4 of System 4

Robert Campbell, April 2011

Introduction to Membrane Processes & Signaling Cascades

Animal cells are enclosed in a phospholipid bi-layer membrane. The 'head' of a phospholipid is hydrophilic (attracted to water), while the hydrophobic 'tails' are repelled by water and are forced to aggregate toward the center between th two heads. The hydrophilic head contains the negatively charged phosphate group, and many contain other polar groups. The hydrophobic tail consists of fatty acid hydrocarbon chains. The hydrophobic tails line up toward each other, forming a membrane with hydrophilic heads on both sides facing the water inside and outside the cell membrane. This mosaic of lipid cell membrane can act as a solvent for many other substances and proteins embedded within it so that they can migrate laterally over the membrane.

The many proteins and other substances embedded, attached, or adjacent to the cell membrane play essential roles in initiating chemical signaling cascades within the cell. Some act as chemical pumps to move potassium, sodium, calcium, and hydrogen ions against a concentration gradient. Figures 2 and 3 illustrate typical arrangements of a great diversity of membrane related chemicals.

Fig. 1

The outer leaflet consists predominantly of phosphatidylcholine, sphingomyelin, and glycolipids. The inner leaflet contains phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. Cholesterol is distributed in both leaflets and adjusts the fluidity of the membrane. The phosphate heads have a net negative charge. Many proteins and other chemicals are also embedded as below.

Fig. 2 [Above]

Some membrane proteins penetrate only part of the way through the membrane. Peripheral membrane chemicals attach to other proteins and do no penetrate.

Fig. 3 [Above]

A few simplified examples of signaling pathways:

Calcium ions (Ca2+) previously pumped into and stored into the ER are rapidly released when Inositol triphosphate (IP3) opens the channel. There are over 300 kinds of transport channels, many acting as pumps to move ions and other chemicals against a concentration gradient.

Fig. 4 [Above]

Steroid hormones derive from lipids and phospholipids mainly in the gonads and adrenal glands. They are lipid soluble and can pass through the membrane to receptors in the cell. Protein hormones such as insulin and growth hormone, as well as monoamine hormones such as adrenaline, cannot pass through the membrane. They must bind to a membrane receptor to initiate a signaling cascade to the nucleus.

Fig. 5 [Above]

G proteins communicate signals from many hormones, neurotransmitters, and other signaling factors. The chemical signals are called ligands when they dock on a receptor. The ligand binding employs electronic forces, not covalent chemical bonds and docking is reversible.

When signal molecules dock on a receptor domain outside of the cell, it causes the embedded protein receptor to alter shape in an intracellular domain that activates a "peripheral" G protein at the inner membrane. The G protein activates a cascade of further compounds, and finally causes a change downstream in the cell.

G protein complexes bind to phosphate groups. G proteins (guanine nucleotide-binding protein family) respond to many signaling factors to activate signaling cascades downstream. They function as molecular switches. When attached to a complex with three phosphate groups (Guanosine triphosphate GTP), they turn on. When they are attached to a complex with only two phosphate groups (Guanosine diphosphate GDP), they turn off.

G proteins regulate metabolic enzymes, ion channels, transporters, and other parts of the cell machinery, controlling transcription, motility, contractility, and secretion, which in turn regulate systemic functions such as embryonic development, learning, memory, and homeostasis. PKA is a protein kinase that is activated by cyclic adenosine mono-phosophate (cAMP).
Cyclic AMP (adenosine monophosphate) is a second messenger, used for intracellular signal transduction, such as transferring the effects of hormones like glucogon and adrenaline, which cannot pass through the cell membrane. It is involved in the activation of protein kinase enzymes (PKA) that regulate the effects of adrenaline and glucagon. It also regulates the passage of Ca2+ through ion channels. cAMP is synthesized from ATP on the inner side of the plasma membrane.
Fig. 6

Phosphorylation

Phosphorylation is the addition of a phosphate (PO4) group to a protein or other organic molecule and plays a critical role in cell signaling. (The agent of exchange is commonly Adenosine Tri-Phosphate (ATP) which can be reduced to ADP or AMP.) It activates or deactivates a great many protein enzymes, including some causing or preventing diseases such as cancer and diabetes. Each amino acid in protein contains a side chain, which distinguishes it from other amino acids. Phosphates are negatively charged so that their addition to a protein will change its characteristic shape through ionic (electronic) forces. Enzymes called kinases (phosphorylation) and phosphatases (dephosphorylation) are involved in switching other enzymes on or off by changing their shape. Within a protein, phosphorylation can occur on several amino acids, usually serine, threonine, and tyrosine. Histidine and aspartate phosphorylation sometimes occurs as part of two component pathways (analagous to stimulus-response to the environment common in prokaryotes). It is relatively uncommon in eukaryotic pathways.

There are thousands of different kinds of proteins in a cell and up to one half of them may be phosphorylated in some cellular state. Phosphorylation often occurs on multiple distinct sites on a given protein to alter its shape. Upon the deactivating signal, the protein enzyme becomes dephosphorylated again and stops working. This is a global mechanism in complex signaling cascades.

Phosphorylation and Energy

Adenosine tri-phosphate (ATP) is the "high-energy" exchange medium in the cell. It is synthesized in mitochondria organelles by adding a third phosphate group to ADP (adenosine di-phosphate) in a process called oxidative phosphorylation. ATP is also synthesized by glycolysis that converts glucose into pyruvate, releasing free energy to form ATP and NADH (reduced nicotinamid adenine dinucleotide). Glucose is synthesized by solar energy in plant cells. Glycolysis in the cytoplasm produces pyruvate which is employed by mitochondria to convert the bulk of the energy originally in glucose to ATP via the citric acid (Krebs) cycle.

Figure 7 shows a couple of steps employing enzymes in a more complex process of energy conversion. Enzymes are proteins that fold into a highly specific shape. Various molecules are attracted and fit in such a way that they are brought into intimate contact and can bond together much more rapidly and accurately than they would at random. Enzymes are highly specific catalysts tailored to each chemical reaction. Enzymes can be turned on and off by attaching or removing phosphate groups. In some cases, other molecules can attach to turn them off or turn them back on by their release in a reverse manner to phosphate groups.

[image]
Fig. 7

Phosphorylation of sugars, such as glucose, is often the first stage of their catabolism in cells because the phosphate group prevents them from diffusing back outside through the membrane transporter. Catabolism is a metabolic process where complex organic compounds are broken down into simpler compounds, for instance, converting glucose into carbon dioxide and water with the release of energy into ATP. Anabolism is the metabolic steps requiring energy to make complex compounds, such as proteins, by assembling simple compounds such as amino acids.

Figure 8 below gives a general picture of how the process works to provide the energy needed by cells to operate.

The adding and subtracting of a phosphate to ADP is a metabolic process. Metabolic processes can be separated into two phases; catabolism is the process of breaking down (breaking down food to make ATP), and anabolism is the process of building up (using the energy created in converting ATP to ADP to build up cells or move molecules around the cell). (CoQ10 is a coenzyme.)
Fig. 8

Endocrine, Autocrine, Paracrine, and Juxtacrine Signaling

Endocrine signaling cascades are initiated by chemical messengers released from the endocrine glands. They circulate in the blood stream and bind to chemical receptors embedded in the membranes of various cells throughout the body that, in turn, initiate a series of events that results in a signaling cascade to the nucleus. Chemical messengers can also inhibit signaling cascades or the cascade may depend on their concentration or other factors.

Autocrine signaling releases a chemical messenger to the outside of a cell that binds to a receptor in the same cell to initate or modify a signaling cascade in that cell.

Juxtacrine signaling docks a signaling chemical directly with the chemical receptor on an adjacent cell. The signaling chemical remains attached to the sending cell. An example of juxtacrine signaling is shown in the "Notch" signal represented above right in Figure 9.

Paracrine signaling releases chemical messengers that rapidly degrade or are reabsorbed over very small distances, such as at synaptic junctions between neurons.

Fig. 9

Other Signaling Mechanisms

Because calcium ions (Ca2+) are an important "second messenger" that affects all cell signaling, it is essential to keep concentrations very low in the cytosol so as not to interfere with other signaling cascades unless it is needed rapidly. PMCA is a membrane transport protein that employs ATP to move calcium against a steep concentration gradient to the outside of the cell or into organelles where it is sequestered for rapid release when needed. Calcium ions also bind with protein buffers that shape enzymes while reducing the amount of free calcium in the cytosol. When calcium is bound with the small buffer calmodulin, transport is accelerated. Sodium-Calcium Exchanger pumps (NCX) are needed to remove calcium ions faster, especially in excitable neuron and muscle cells. Calcium has a vital and ubiquitous influence on cell signalling of all kinds in cells of all kinds.

Sodium-potassium (Na+K+) pumps move these two ions in opposite directions across the plasma membrane to maintain a membrane resting potential. Three sodium ions are moved out by hydrolyzing ATP and two potassium ions move in. Potassium can leak back out while sodium ions cannot leak in. The sodium concentration outside the cell membrane provides the driving force for several secondary active transporters which import glucose, amino acids, and other nutrients into the cell as illustrated in Figure 10. Another important function of the Na+K+ pump is to maintain the volume of the cell. Most proteins inside the cell are negatively charged and collective positive ions around them. Osmosis of water into the cell would cause it to swell unless checked.

The membrane protein pump transporter can also relay extracellular signaling into the cell. The downstream phosphorylation events include the activation of mitogen-activated protein kinase (MAPK) signal cascades, mitochondrial reactive oxygen species (ROS) production, as well as activation of phospholipase C (PLC) and inositol triphosphate (IP3) receptor (IP3R) in different intracellular compartments. Hydrogen peroxide and nitric oxide are also important in cell signaling.

Signal peptides are amino acid sequences that direct proteins (synthesized in the cytosol) to organelles such as the nucleus, mitochondria, endoplasmic reticulum (ER) and peroxisomes. A Signal Recognition Particle (SRP) shuttles between the ER and the cytosol and binds to the signal peptide on the leading end of the peptide string as soon as it is synthesized by a ribosome to chaperone the ribosome to the ER. The SRP detaches when the signal peptide guides the protein string to thread inside through the ER membrane translocator pore as it is assembled. Most ER signal peptides are relayed on to the Golgi Apparatus. A nuclear localization signal (NLS) is a peptide directing cytosol protein to the nucleus. The nucleolus is targeted by a nucleolar localization signal (NOS). The mitochondrial targeting signal (MTS) directs to mitochondria. PTS1 and PTS2 direct to peroxisomes.

Gap junctions are protein pores joining some cells in direct contact. This allows some small molecules and ions to diffuse from one cell to another. Gap junctions can be opened or closed by internal conditions in the cells depending on circumstances.

Some small molecules and ions can passively diffuse through the membrane by processes such as osmosis. Other membrane transport mechanisms involve endocytosis and exocytosis as reviewed later.

Active transport of sodium and potassium ions (right) powers secondary active transport of glucose, amino acids, and growth factors stored in the extracellular matrix where protease enzymes break down some proteins into amino acids essential for protein synthesis in the cell.
Fig. 10

Complex Multi-Component Signaling Pathways

Complex multi-component signaling pathways provide opportunities for feedback, signal amplification, and interactions inside one cell between multiple signals and signaling pathways. For example, many growth factors bind to receptors at the cell surface and stimulate cells to progress through the cell cycle and divide. Several of these receptors are kinses that start to phosphorylate themselves and other proteins when binding to a chemical messenger or ligand. This phosphorylation can generate a binding site for a different protein and thus induce protein-protein interaction.

An example is given in Figure 11. The ligand (epidermal growth factor EGF) binds to the receptor called EGFR. This activates the receptor to phosphorylate itself (see the small P attached). The phosphorylated receptor binds to an adapter protein (GRB2), which couples the signal to further downstream signaling processes. For example, one of the signal transduction pathways that are activated is called the mitogen-activated protein kinase (MAPK) pathway. The MAPK protein is an enzyme, a protein kinase that can attach phosphate ions to target proteins such as the transcription factor MYC and, thus, alter gene transcription and, ultimately, cell cycle progression. Many cellular proteins are activated downstream of the growth factor receptors (such as EGFR) that initiate this signal pathway.

Epidermal growth factor EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein-tyrosine kinase activity of the receptor to phosphorylate. This initiates a signal transduction cascade (via GRB2) that results in a variety of biochemical changes within the cell - a rise in intracellular calcium levels, increased glycolysis and protein synthesis, and increases in the expression of certain genes, including the gene for EGFR - that ultimately lead to DNA synthesis and cell proliferation.

Activated Ras activates the protein kinase activity of RAF kinase. RAF kinase phosphorylates and activates MEK. MEK phosphorylates and activates a mitogen-activated protein kinase (MAPK). RAF, MEK, and MAPK are all serine/threonine selective protein kinases.

In the technical sense, RAF, MEK, and MAPK are all mitogen-activated protein kinases. MAPK was originally called "microtubule-associated protein kinase" (MAPK). One of the first proteins known to be phosphorylated was a microtubule associated protein (MAP). Many additional targets for phosophorylation by MAPK were later found, and the protein was re-named. The series of kinases from RAF to MEK to MAPK is an example of a protein kinase (phosophorylation) cascade. Such series of kinases provide opportunities for feedback regulation and signal amplification.

In simpler terms, the mitogen (EGF) binds to the membrane ligand. This means that Ras (a GTPase) can swap its GDP for a GTP. It can now activate MAP3K (e.g., Raf), which activates MAP2K, which activates MAPK. MAPK can now activate a transcription factor, such as myc.
Fig. 11

In some cases, receptor activation caused by ligand binding to a receptor is directly coupled to the cell's response to the ligand. For example, the neurotransmitter GABA can activate a cell surface receptor that is part of an ion channel. GABA binding to a GABA A receptor on a neuron opens a chloride-selective ion channel that is part of the receptor. GABA A receptor activation allows negatively-charged chloride ions to move into the neuron, which inhibits the ability of the neuron to produce action potentials. However, for many cell surface receptors, ligand-rreceptor interactions are not directly linked to signaling or to the cell's response. The activated receptor must first interact with other proteins inside the cell before the end physiological effect of the ligand on the cell's behavior results. Often, the behavior of a complex chain of interacting cell proteins is altered following receptor activation.

Some signaling transduction pathways respond differently depending on the amount of signaling received by the cell. For instance, the hedgehog protein activates different genes, depending on the amount of hedgehog protein present.

In summary, complex multi-component signal transduction pathways provide opportunities for feedback, signal amplification, and interactions inside each cell between multiple signals and signaling pathways.

Some signaling molecules can function as both a hormone and a neurotransmitter. For example, opinephrine and norepinephrine can function as hormones when released from the adrenal gland and are transported to the heart by way of the blood stream. Norepinephrine can also be produced by neurons to function as a neurotransmitter within the brain. Estrogen can be released by the ovary and function as a hormone or act locally via paracrine or autocrine signaling. Active species of oxygen and nitric oxide can also act as cellular messengers. This process is dubbed redox signaling.

Complex Insulin Signaling Pathways

A few of the insulin signaling pathways are shown in Figure 12. They include the regulation of glucose and lipid metabolism, cell growth, differentiation, and general gene expression.


Fig. 12

Endocytosis and Exocytosis

Membrane transport systems such as diffusion and pumps are not sufficient to meet all of the needs of cells. Endocytosis is needed to ingest needed raw materials in the required quantities and exocytosis is needed to exort cell products and dispose of waste. The general methods of endocytosis are illustrated in Figure 13.

Phagocytosis is facilitated by phagocyte surface receptors that attach to ligand coated particles such as nutrients, microorganisms, and proteins. In the immune system, it is a major mechanism used to remove pathogens and cell debris. Bacteria, dead tissue cells, and small mineral particles are all examples of objects that may be phagocytosed. The resulting phagosomes eventually fuse with lysomes which contain over 40 kinds of enzymes that digest the contents for use in the cell and discharge waste products by exocytosis.

Pinocytosis usually occurs at highly ruffled regions of the plasma membrane. The resulting vesicles capture extracellular fluid, molecules, and dissolved food in a non-specific manner. The fluids captured include all solutes present. The cell engulfs already dissolved or broken down food. They fuse with other vesicles such as endosomes and lyosomes.

Receptor mediated endocytosis occurs at coated pits on virtually all cells such that the resulting vesicle has a specific protein coat that identifies it. The coat may be made up of a complex of clathrin proteins in the cytosol. Coated pits can concentrate large extracellular molecules that have different receptors for many ligands such as low density lipoprotein (LDL), transferrin (iron uptake), growth factors, antibodies and many others. Receptor mediated endocytosis is also actively implicated in transducing signals from the cell periphery to the nucleus.

Caveolae are another type of coated membrane cave-like buds which exist on the surface of many, nut not all, cell types. They have a cholesterol-binding protein caveolin with a membrane enriched in cholesterol and glycolipids. They can constitute up to a third of the plasma membrane area of some cell tissues, such as the smooth muscle of the gut. The uptake of extracellular molecules is specifically mediated via receptors in caveolae.


Fig. 13

Many cells in the body use exocytosis to release enzymes or other proteins such as signaling molecules that act in other areas of the body. For instance, clusters of α- and β-cells in the islets of Langerhans in the pancreas secrete the hormones glucagon and insulin, respectively. These enzymes regulate glucose levels throughout the body. As the level of glucose rises in the blood, the β-cells are stimulated to produce and secrete more insulin by exocytosis. When insulin binds to liver or muscle, it stimulates uptake of glucose by those cells. Exocytosis from other cells in the pancreas also releases digestive enzymes into the gut.

Nerve cells communicate across synaptic junctions by releasing neurotransmitters stored in vesicles near the synaptic cleft. The action potential of a firing neuron signals the vesicle to make contact with the plasma membrane and secrete their contents into the synaptic cleft for th other neuron to receive the chemical messenger. Components of the vesicle and extra neurotransmitter molecules are quickly taken up and recycled by the neuron to form new vesicles that are ready to send another pulse to an adjacent neuron. Neurons need to send many signals each second, which indicates how tight the controls that regulate exocytosis.

An immune cell communicates with a virally infected cell that it must destroy itself to preserve other cells around. An infected cell displays viral by-products on its surface, like turning on red warning lights to attract immune cells. Immune cells, such as the killer T cells, wander through the body. They recognize and position themselves so that their plasma membranes are very close. In a rapid succession, the killer T cells mobilize secretory vesicles filled with enzymes, like perforin and granzyme B, adjacent to the inner side of their plasma membranes. In response to a signal, the vesicles undergo exocytosis and release their contents. These enzymes punch holes in the plasma membrane of the infected cell. This causes the cell to undergo self-destruction or apoptosis, also known as programmed cell death, to prevent further spread of the virus.

Lyosomes are produced by the Golgi Apparatus. They contain a complement of enzymes that digest the contents of phagosomes after they merge. The products, such as sugars and amino acids, diffuse from the fused lyosome for use in the cell and the waste products are removed from the cell by exocytosis.

Membrane fusion requires energy and the interaction of special "adaptor" molecules present on both the vesicle and plasma membrane. The adapter molecules are highly selective and only allow vesicles to fuse with membranes of particular organelles, thus preventing harm to the cell.

Once the appropriate adapter molecules bind to each other (docking), energy stored and released by ATP forms a fusion pore between the vesicle membranes and plasma membrane. The contents of the vesicle are released to the exterior of the cell (or the interior of an organelle such as a mitochondrion or another vesicle) as the fusion pore widens.

The vesicle ultimately becomes part of the plasma membrane or is recycled back to the cytoplasm. Vesicle formation can be very rapid.
Fig. 14

Membrane Merging

Figure 15 illustrates the action of SNARE proteins docking a vesicle for exocytosis. Complementary versions of the protein on the vesicle and the target membrane bind and wrap around each other, drawing the two bilayers close together in the process. Membrane fusion can be induced preferentially by Ca2+ or Mg2+ ions at various threshold concentrations for different phosopholipid species of membrane. The lipid membranes must also be in a fluid state that may be transiently induced by ion concentrations.


Fig. 15

Overall Membrane Dynamics

Endocytosis and exocytosis tend to balance the quantity of plasma membrane. More membrane is also made at the Endoplasmic Reticulum with appropriate protein markers. Membrane embedded chemicals are modified during the course of vesicle migration to and from the Golgi apparatus and in various vesicles. Vesicles receive chemical address markers. Some are moved along the microtubule cytoskeleton by molecular motors such as dynein and kinesin. For example, vesicles carrying neurotransmitters must migrate long distances to their axon terminals, for example, from the ventral horns of the spinal column to the muscles of the big toe. In all of these processes, membrane dynamics must be closely controlled to maintain the required shape of cells.


Fig. 16

The Extracellular Matrix

In animals, the extracellular matrix is composed of protein and polysaccharides. It can provide strength, such as cartilage that prevents joints from grinding against each other. It can provide structural support, such as the bone of skeletons. It can help organize cells, such as keeping tendons attached to bones. It can assist with cell signaling and indicate changes in the environment. It can also act as a storehouse to stockpile materials such as proteins, amino acids, sugars and other substances that may be needed by the cell on short notice.

The major proteins found in the extracellular matrix provide adhesion and structure. Adhesion is achieved by the proteins fibronectin and laminin which attach cells to the extracellular matrix. Structure is achieved by the proteins collagen and elastin which allow some body parts to stretch and return to the original shape, such as skin and inhaling air into the lungs.

The major polysaccharides in the extracellular matrix are glycosaminoglycans (GAGs) (or proteoglycans). GAGs are very long chains of disaccharides. They tend to be negatively charged and thus get along well with water. As part of the extracellular matrix, they take on a gel-like consistency, which makes them good for uses such as joint fluid, and they are also found in the skin and eyes. Proteoglycans may also help to trap and store growth factors and other materials needed in the cell on short notice. In some cells, the extracellular matrix is thus like a storage yard.

Collagen is a group of proteins found exclusively in animals, especially in the flesh and connective tissues of mammals. It is the main component of connective tissue, and is the most abundant protein in mammals making up about 25% to 35% of the whole-body protein content. About 30 kinds of collagen have been identified. In the form of elongated fibrils, it is mostly found in fibrous tissues such as tendon, ligament, and skin. Other forms are abundant in cornea, cartilage, bone, blood vessels, the gut, and inter-vertebral discs.


Fig. 17

Summary of Membrane and Signaling Details

From the above review of cell membrane and signaling processes, a few key distinctions may be made, despite the great complexity involved. They may be summarized as follows:

1.