Cellular Signaling Notes

(Move to Outline HERE)

Introduction

Multicellular organisms have challenges not faced by unicellular ones. A very important one is the need for cells in different parts of the body to signal and respond to signals from other parts of the body (see HERE). A good deal of the signaling is accomplished via receptors that are in the membranes of cells. Membrane receptors bind to specific ligand molecules and initiate a molecular response that alters the catalytic activities occurring inside of cells. Though some signaling molecules travel ACROSS the cellular membrane and exert their effects directly inside of cells, most of them have actions mediated through receptors. The focus of this chapter is how information is transmitted from outside of a cell to the inside. This process is called signal transduction. The inability to respond properly to organismal signals is harmful and can, in many cases, lead to cancer.

System Organization

The examples I will go through relate to ligands found OUTSIDE the cell, that bind to a cellular receptor, and that initiate a cellular response INSIDE the cell. Molecules that cross the cellular membrane barrier and cause responses directly will not be discussed here.

Ligands that are made by the body for signaling are called primary messengers, since they are released into the bloodstream in response to a bodily need and aim to initiate a response. A very good example is insulin, which is a small protein that is released from the pancreas in response to increasing blood sugar after consumption of a mean. Specific cellular receptors bind primary messengers and transmit information inside of the cell without the ligand ever getting into the cell. If we think of a house as an analogy for a cell, your telephone is analogous to the receptor. A person outside of your house initiates a signal that results a receptor signal (ringing phone). When you answer the phone, the signal of the "ligand" (person making the call) is communicated to you INSIDE the house. Your response may be to clean the house, etc., in response to what the person on the outside tells you.

For cells, the "telephone line" is a protein embedded in the cellular membrane called a membrane receptor. Membrane receptors project through BOTH sides of the cellular membrane. The dialing/ringing of the phone by your friend on the 'outside' corresponds to a molecule (called a ligand or primary messenger) on the outside of the cell binding to the outer portion of the membrane receptor. This binding causes changes in the shape and/or catalytic properties of the receptor. These changes are manifested on the portion of the membrane receptor at the inside of the cell, which initiates a signal inside of the cell (you taking action, such as cleaning your house). Changes on the receptor on the inner portion of the cell often result in production of a new molecule (called a second messenger) that begins a series of responses inside the cell. A simple schematic of this process is shown HERE. Common second messengers include cAMP, cGMP, calcium, inositol 1,4,5 trisphosphate (IP3), and diacylglycerol (DAG) (HERE). Production of second messengers as a result of enzymatic processes results in amplification of the signal, as a single enzyme can catalyze formation of many second messenger molecules. These second messengers are free to diffuse throughout the cell can cause their effects. Second messengers are used by many signaling systems and this can cause confusion, in some cases.

Protein Phosphorylation

Another step in communicating a signal is that of phosphorylation of proteins. This can occur as a result of binding of a second messenger to a protein kinase, causing it to be activated or by direct activation of a membrane receptor. Proteins can be phosphorylated readily on their threonine, serine, or tyrosine hydroxyl groups. Reversing the signal of protein phosphorylation (removal of phosphate from proteins) requires action of enzymes called protein phosphatases. It is important that signals that are turned on also be turned off when appropriate. Failure to turn of a signal, for example, that tells a cell to proliferate, can lead to uncontrollable growth and ultimately a tumor.

Membrane receptors have a common structural motif that consists of seven helices that project through the cellular membrane (see HERE and HERE). For this reason, they are called (as a group) seven transmembrane membrane helix receptors, or 7TM receptors, for short. Two such 7TM receptors are 1) rhodopsin, which responds to photons instead of chemical ligands and 2) the beta-adrenergic receptor (HERE), which responds to the chemical ligand epinephrine (also called adrenalin). Binding of a ligand to the beta-adrenergic receptor or interaction of a photon with rhodopsin has the same effect - a change in configuration of the part of the protein on the inner part of the cell. These structural changes are the key to the actions these proteins initiate.

In the case of the beta-adrenergic receptor, binding of epinephrine to the receptor's outer portion causes the inner portion to activate a protein called a G protein (because it binds guanine-containing nucleotides). (Note - all 7TM receptors appear to be linked to various G proteins). This is the first step in the intracellular process of signal transduction. G proteins have two states - activated and inactivated. In the inactivated state, G proteins are bound to GDP. This form of the G protein has three subunits (alpha, beta, and gamma - shown HERE). Ligand binding by the beta-adrenergic receptor causes the receptor (on the inside of the cell) to stimulate the replacement of GDP by GTP in the alpha subunit. This, in turn, causes the alpha subunit to dissociate from the beta and gamma subunits. The freed alpha subunit then interacts with an enzyme known as adenylate cyclase (HERE), which catalyzes formation of cAMP (second messenger). Thus, binding of epinephrine to the beta-adrenergic receptor ultimately causes cAMP to be produced. This has many effects, as will be seen later in the term and below. There are MANY forms of alpha, beta, and gamma subunits of G proteins, making thousands of possible combinations possible.

Deactivating G Proteins

Remember that it is important for cells to turn off signals, as well as turn them on. In the case of G proteins, this is accomplished by an intrinsic GTPase activity in G alpha units (HERE). Thus, after they get GTP, they use it for seconds to minutes before they break it down to GDP. When GDP is thus formed, the G alpha then re-binds the beta and gamma subunits, leaving it inactivated. The receptor itself can be deactivated by 1) unbinding of ligand and 2) phosphorylation of the receptor by a receptor kinase (HERE) and subsequent binding by the protein called arrestin, which also helps release the ligand.

cAMP Effects

cAMP is a second messenger and propagates the message for many signaling pathways. It accomplishes this by acting mostly on a single enzyme - protein kinase A (PKA). PKA has four subunits - two regulatory subunits (r) and two catalytic subunits (c). In the absence of cAMP, they form a complex as r2c2 that is inactive. cAMP binds to the r subunits, freeing the catalytic subunits, which become active. The active PKA subunits, in turn, can catalyze phosphorylation of many different proteins. For example, enzymes of glycogen synthesis are turned off by phosphorylation, wherease enzymes of glycogen breakdown are turned ON by phosphorylation.

Other effects active PKA subunits can cause via phosphorylation also include 1) activation of transcription by phosphorylation of a transcription factor (cAMP-response element binding (CREB) protein) and 2) closing potassium channels of neurons.

Phospholipase C and the Phosphoinositide Cascade

Other 7TM receptors act to activate an enzyme called phospholipase C through action of another G protein called Galpha-q. Activated phospholipase C cleaves a molecule found in membranes - phosphatidyl inositol 4,5 bisphosphate (PIP2) to yield TWO messengers - inositol 1,4,5 trisphosphate (IP3) (HERE) and diacylglycerol (DAG) (HERE). IP3 can diffuse away from the membrane, but DAG remains associated with the membrane (HERE). IP3 works by releasing Ca++ from intracellular stores (such as the endoplasmic reticulum or sarcoplasmic reticulum) of this ion. Calcium released in this way can stimulate muscular contraction and glycogen breakdown. IP3 can be broken down by phosphatases or turned into inositol. Lithium may act on bipolar disorder by inhibiting breakdown of IP3. DAG acts to stimulate another protein kinase (protein kinase C) that, in turn, phosphorylates many proteins. DAG is rapidly metabolized, as well, - either hydrolysis to yield glycerol and fatty acids or phosphorylation to form phosphatidic acid.

Calcium as a Cytosolic Messenger

Calcium is kept at artificially low levels in cells compared to their surrounding fluid. This is due, in part, to the problems associated with high calcium concentration - precipitation of molecules like DNA can occur in too much calcium. The levels are kept WAY lower than necessary, however, and cells use this concentration difference across the cell membrane as a way of rapid signaling. Opening channels to let calcium into cells can bring a tremendous amount of calcium into the cell in a very short time period. When calcium is free in the cell, it can bind tightly to proteins, causing them to change conformation and, as a result, activity. One protein that binds to calcium and helps it to exert its effects is calmodulin (HERE). Calmodulin has a structural feature called EF Hand (HERE) that binds calcium. When calmodulin binds calcium, it changes conformation (HERE), exposing hydrophobic residues that can, in turn, interact with other proteins. Consequently, binding of calcium by calmodulin causes both the calmodulin to change and (more importantly) other proteins to be affected as a result of calmodulin binding. Some of the proteins affected by calmodulin include (surprise!) protein kinases, called CaM kinases that phosphorylate other proteins.

I will refer you to the book for the remainder of this chapter.