EFB325 Cell Physiology

Hormones and cell-to-cell signaling

Multicellular organisms have tissues and organs with differentiated cells that perform specialized functions important for the homeostasis of the organism. The organization of these tissues through development and the day-to-day function of these specialized cells and tissues must be coordinated especially when responding to particular stresses, stimuli, or nutritional conditions. This coordination must occur over long distance and may involve a diverse set of responses in different cell types.

Type of cell signaling

• contact dependent=direct cell-to-cell interactions, important through development
• neuronal=fast electrical signals, converted to neurotransmitter chemical signals at termini
• paracrine hormone=local chemical signaling, within a few cells distance
• endocrine hormone=chemical signals produced in specialized cells, then distributed long-distance through the bloodstream

Hormones can affect blood sugar balance, heart rate, blood pressure, blood clotting, inflammation, growth and development, and reproductive cycle; different cells must respond differently and particular cells must respond to only a subset of the signals sent

What are the hormone molecules and where are they synthesized?

• chemical messengers secreted by one cell type in order to regulate the function of various other cells
• hormones are compounds that are not normal metabolic intermediates-they are special molecules
• generally have a short lifetime, so that levels can be regulated over short time periods
• most are water-soluble (hydrophilic)-are derivatives of amino acids, a fatty acid (arachidonic acid) or are peptides/proteins
• steroid hormones are hydrophobic, so they are carried in the bloodstream by carrier proteins
• hormones are very dilute in the bloodstream, so they must be able to act at very low concentrations
• endocrine hormones are produced and secreted by glands, such as: thyroid gland, thymus gland, adrenal glands, pancreas, gonads
• paracrine hormones are very short-lived and are produced by a cell to act only on local cells

How are hormones perceived by cells and then how is that signal converted into a response by the cell?

Hormones are received by receptor proteins in cells, initiating a response cascade

The receptor and subsequent elements of the response are together called a signal transduction cascade

Hormone receptors

• binding of a hormone (generically called the ligand) to a receptor protein is very much like substrate-enzyme binding; the receptor has a highly specific binding site
• since hormones are present at low concentrations, then the receptor must have a high affinity for the ligand (the hormone) (they have a low K_d)
• when the ligand (the hormone) binds, the receptor becomes activated and initiates the response cascade

Features of signal cascades:

• they relay the signal from the site of reception to the site of response within the cell
• they transform the signal from its original form to a form that can stimulate the response
• they usually amplify the signal from a few molecules originally to a large cellular response
• they can distribute the signal, stimulating diverse responses by multiple cellular components
• they allow the response to be modulated and regulated including interactions with other simultaneous signaling cascades

Cell sensitivity to a hormone

• a cell's sensitivity to a hormone is determined by the receptors it contains; including the number of a particular receptor protein, the affinity of the receptors to bind hormone, or the ability of the receptors to become activated upon hormone binding
• a cell can change its sensitivity, generally to become less sensitive after constant exposure to a particular hormone usually by reducing the number of receptor proteins (this is a way that the cell can adapt to a condition)

Hormone receptors are either intracellular or cell-surface (plasma membrane) receptors

• steroid hormone receptors are intracellular, since the steroids can pass through the membrane
• hydrophilic hormones must bind to the receptor on the outside of the cell

Steroid hormone receptors generally function by activating the transcription of a particular gene or genes

• these intracellular receptor proteins can be located either in the cytosol or in the nucleus
• upon binding hormone, steroid hormone receptors become active as transcription factors
• the gene turned on by the hormone:receptor complex often produces a protein that acts as a transcription factor itself and turns on many other genes (thus the signal is amplified)

Nitric oxide (NO) can pass into cells and activate enzymes directly

• NO is a gas that is produced in the cells that line the blood vessels and it can diffuse into nearby smooth muscle cells in the walls of the blood vessels
• it binds directly to a regulatory enzyme and triggers smooth muscles to relax, thus increasing blood flow through those vessels
• the drug nitroglycerine is converted to NO and triggers relaxation of the coronary blood vessels to relieve angina
• release of NO is also responsible for triggering increased blood flow in the penis - Viagra functions to prolong the NO signal

There are three types of cell-surface (plasma membrane) receptors: ion-channel-linked receptors, G-protein-linked receptors, and enzyme-linked receptors

• binding of the hormone on the outside of the cell causes a change in activity of the receptor protein, usually on the cytoplasm side of the plasma membrane
• responses to hormones can be rapid, involving direct alteration of the activity of existing enzymes or slower, involving activation of genes that must be transcribed, translated, and folded
• the response cascade involves a series of components that are switched on or off
• this switching often involves phosphorylation by a kinase and dephosphorylation by a phosphatase
• in fact, kinases can phosphorylate and activate other kinases, setting up phosphorylation cascades (there is almost always a phosphatase to counteract the activity of the kinase)
• G proteins are switched on when they bind GTP, then switched off when the GTP is hydrolyzed to GDP

Ion-channel-linked receptors act in nerve signaling to convert a chemical signal to electrical

• when they bind neurotransmitters, the ion channel portion of the protein is triggered to open, generating a change in membrane potential

G-protein-linked receptors activate a G protein, which can diffuse to activate regulatory enzymes

• upon binding ligand on the outside of the cell, the receptor changes conformation and activates a G protein on the inside of the cell
• G protein= guanidine nucleotide-binding protein (GTP or GDP-binding protein)
• G proteins have three subunits, a, b, and g (three different subunits=heterotrimeric protein)
• the a subunit binds the GTP or GDP and has GTPase activity that can hydrolyze GTP back to GDP
• all G-protein-linked receptors have similar structure, with a domain on the outside with a binding site for the hormone; 7 membrane-spanning a-helices; and a domain on the cytosolic side that interacts with a specific G protein
• different receptors can activate different types of G proteins

The activation/inactivation scheme

• the receptor binds ligand
• a change in shape of the receptor causes the associated G protein to release GDP and bind GTP (GDP/GTP bind to the a subunit)=this activates the G protein
• the a subunit dissociates from the receptor and from the b/g complex
• a and/or b/g complex activate target enzymes
• after a short time, the a subunit hydrolyzes GTP to GDP, then reassociates with the b/g complex and the G protein is inactivated

G proteins activate target enzymes

The activated G protein can now distribute and amplify the signal to functional or regulatory enzymes in the cell. It may activate a important functional protein directly, such as triggering an ion channel to open, but commonly G proteins activate specific target enzymes that produce many additional small signaling molecules called second messengers (the hormone was the first messenger). Second messengers can activate broad and diverse target proteins in the cell in the process of stimulating the response to the hormone.

G proteins can activate either of the enzymes:  adenylate cyclase or phospholipase C

Adenylate cyclase is an enzyme that produces the second messenger cyclic AMP (cAMP)

• adenylate cyclase converts ATP to cAMP, which is a rare compound in the cell
• cAMP is required by a specific protein kinase called cAMP-dependent protein kinase (also called protein kinase A or PKA)
• when cAMP is present, PKA is active and it then phosphorylates a variety of enzymes, which can activate or inactivate those enzymes-those enzymes will cause the change in cell metabolism which is the response
• cAMP is degraded by phophodiesterase to AMP (the signal transduction chain then is no longer active)

Examples of cAMP signaling (commonly involved in response to adrenaline)

• in skeletal muscles, increased cAMP causes glycogen breakdown, releasing glucose
• in cardiac muscle, increased cAMP increases heart rate
• in smooth muscle, increased cAMP inhibits contraction
• in fat cells, increased breakdown of triglycerides, releasing acetyl-CoA
• in intestinal epithelial cells, increased cAMP causes increased secretion of salts and water to the lumen of the intestine
• caffeine is a phophodiesterase inhibitor, it prevents the degradation of the low levels of cAMP, thus cAMP levels stay high-resulting in increased heart rate and increased secretion of water in the intenstine
• cholera toxin inhibits the GTPase activity of the alpha-subunit of the G-protein, so that it remains active, thus adenylate cyclase remains active, cAMP levels remain high, and the intestines secrete too much water, causing dehydration

Phospholipase C is an enzyme that sends a signal resulting in increased levels of the second messengers DAG and IP₃

• phospholipase C cleaves a particular type of phospholipid into two molecules, inositol triphosphate (IP₃) and diacylglycerol (DAG)
• IP₃ diffuses to the ER membrane and triggers Ca²⁺ channels in the ER to open releasing Ca²⁺ into the cytosol
• DAG diffuses along the PM and together with Ca²⁺ can activate protein kinase C (PKC), which then activates other functional enzymes to cause the response
• 4 Ca²⁺ atoms also bind to the protein calmodulin (CaM), and the Ca²⁺:calmodulin complex can activate calmodulin-dependent protein kinases (CaM kinases) which can activate other functional enzymes to cause the response

Enzyme-linked receptors

Most commonly these are receptor tyrosine kinases, which are important in growth response to growth factor hormones

• bind the ligand on the outside of the cell, this brings two receptor subunits together, which activates a tyrosine kinase on the inside
• single polypeptide with one membrane-spanning -helix (remember G protein-linked receptors had 7 membrane spanning domains) has a ligand (hormone)-binding domain on the outside of the cell; inside the cell, there is the tyrosine kinase domain
• the active kinase phosphorylates tyrosines within the receptor polypeptides themselves (they are autophosphorylated)
• other proteins bind, can be phosphorylated, and are activated

Epidermal growth factor (EGF) and the Ras pathway

• EGF and other growth factors are paracrine hormones secreted to stimulate cell division, for example to replace cells destroyed in a wound
• EGF binds to and activates a tyrosine kinase receptor
• the tyrosine kinase activates an adaptor protein, which activates a Ras-activating protein
• the Ras protein is a type of G protein with only one subunit (it is like the a subunit by itself )
• the Ras-activating protein causes Ras to release GDP and bind GTP, which activates Ras
• once active, Ras then activates a cascade of protein kinases, which phosphorylate and change the activity of transcription factors, which activates the transcription of genes involved in cell growth and division, stimulating cell division

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