Cell biology concepts and applications

G Protein-Linked Receptors

An organism must respond appropriately to its internal and external environments day after day in order to survive. The organism's cells respond to internal and external stimuli much like tiny computers that process numerous inputs and also produce numerous outputs in daily existence (Kennedy 2004). These stimuli are signals that come from the general environment or the cells of other or co-existing organisms, proximate or distant, and this exchange of stimuli and responses involves three sequential processes. These are signal that binds to the receptor protein, the binding that sends a message to the receiving cell's cytoplasm that amplifies it, and the receiving cell's change or response to the signal (Kennedy).

Cells must process the perceived information from the environment and form appropriate responses to it and not all cells can do this. In order to interpret signals, a cell should have the appropriate receptor protein (Kennedy 2004). Multi-cellular organisms possess the genetic information for all receptor proteins, but because there are differential gene expressions, different cells have different receptors. Signals, whether as chemical molecules or physical stimuli, and their interpretation constitute the order called life and these signals are used from the earliest stage of embryonic development to the death of the entire organism (Kennedy). They provide information to the cells of multi-cellular organism within a tissue, organ or the entire body, such as in wound healing, cell replacement or death, the moment-to-moment maintenance of sufficient and appropriate concentrations of nutrients and numerous other activities on the cellular to organic levels (Kennedy). This process requires a receptor, transduction and effects and cells possess specific receptor proteins for interacting to specific signals. Signal transduction is the change of a signal from one form to another and numerous transductions simultaneously occur through a pathway by means of blood circulation. A receptor changes form when binding and conforming to its specific signal molecule and, as a result, exposes a protein kinase (Kennedy). Protein kinases are the common intermediary agents in signal transduction.

A receptor is genetically determined and a cell does not respond to all the signals or stimuli it receives (Kennedy 2004). A ligand is the signaling molecule that binds the receptor and the receptor binds the ligand according to chemistry's law of mass action.

That specific receptor, called seven-spanning G. protein-linked receptor, lies at the beginning of a modular-type system of information transfer (Kennedy), which consists of a receptor that spans the plasma membrane, a G. protein, and an effector protein. G proteins are a binding location for the G. protein-linked receptor and a nucleotide called GDP/GTP (Kennedy). G proteins are active when bound to GTP and inactive when bounds to GDP.

Signal transduction is quite regulated. Cells must often revert to previous states and need to regulate the transduction mechanism (Kennedy 2004). And in order to remain responsive to stimuli, cells must quickly restore themselves into the previous state. Signaling pathways are like switches of sophisticated electrical systems where many complex cellular changes develop or are formed from the interactions of many simple switching systems (Kennedy).

The review of G. protein signaling was published almost two decades ago and central to this signaling process have been cell surface receptors (Morris and Malbon 1999). But since then, almost 20 heterotrimeric G. proteins and different groups of effect units, such as adenylyl cyclasses, that detail the physiological aspects of signaling through the given pathway, found continue to engage the interest and fascination of medical research. It is, therefore, the objective of this paper to attempt at grasping the fundamentals of the large and complex body of information already collected and still in progress on the subject. It will highlight the basic nature of G. protein-linked signaling and how physiological regulation occurs through particular mechanisms (Morris and Malbon).

DISCUSSION

Cells in multi-cellular organisms, like animals, need to communicate among themselves in directing and regulating growth, development and organization (Altruis Biomedical Network 2003). Such communication modes include secreting chemicals that signal to distant cells, display cell surface chemicals that influence other cells in direct physical contact, and directly through porous cellular points called gap junctions (Altruis Biomedical Network). Endocrine signaling demonstrates the first mode, wherein hormones are secreted in the bloodstream to distant target cells. Paracrine signaling illustrates the second mode, wherein local chemical mediators are secreted and act only on cells in the proximate environment. And synaptic signaling exhibits the third mode, wherein molecules are released by vesicles at those junctions called synapses. The molecules are neurotransmitters that spread out and act only on the postsynaptic target cell (Altruis Biomedical Network). Protein receptor molecules that are on or within the target cells bind to the hormone, paracrine or neurotransmitter and a response results, depending on the speed and selectivity of the delivered signal.

Hydrophilic molecules and hydrophobic prostaglandins induce cellular response through specific cell membrane receptors on the target cell (Altruis Biomedical Network 2003). These protein receptors, in turn, bind the signaling molecule, as if in strong affinity, and transduce signals that form or influence a certain cellular behavior. The response of the target cell derives from intracellular second messenger molecules, like cAMP, inositol phosphate and calcium. The three families of cell surface receptors, based on signal transduction mechanisms, are channel-linked receptors, catalytic receptors and G. protein-linked receptors. Channel-linked receptors are transmitter gated ion channels involved in fast synaptic signaling; catalytic receptors are similar to enzymes when activated by a specific ligand; and G. protein-linked receptors, when bound to a specific ligand, indirectly activate or inactivate a separate plasma membrane-bound enzyme or ion channel (Altruis Biomedical Network). This interaction between an enzyme or ion channel and a G. protein-linked receptor is mediated by a GTP. Chemical changes and events within the target cell and usually affect the concentration of secondary intracellular messengers, such as cAMP and inositol triphosphate. These intracellular messengers, in turn, affect the behavior of other intracellular proteins. The effects of these intracellular messengers are quickly reversed with the removal of the extra-cellular signal.

A protein-linked receptors are found in the cell membrane with an extracellular domain and an intracellular domain and the peptide chain always spanning the membrane (Lynn 2004). When binding to the extra-cellular domain, the hormone leads to a conformational change and causes the intracellular domain to activate G. proteins, which can raise the level of enzyme activity or decrease that in the secondary messenger systems (Lynn). There are non-G protein-linked surface receptors too. These have an intracellular domain, which is activated when the hormone binds to the extra-cellular domain. The intracellular domain either goes through an intrinsic enzyme activity itself or activates other enzymes inside the cell. These enzymes are often involved in kinase activity, such as the tyrosine-kinase receptor for insulin (Lynn).

Second messenger systems are activated when stimulated by the receptors and these systems amplify the signal (Lynn 2004). Only one hormone molecule can stimulate one receptor at a time and this stimulated receptor can produce many second messengers, every one of which can, in turn, stimulate other molecules within the cell. A single hormone molecule can, thus create a huge effect and this explains why hormone concentrations in the blood are quite low (Lynn). The main types of second messengers are cyclic adenosine monophosphate or cAMP, IP3 and diacylglycerol or DAG, and calcium. The enzyme adenylate cyclase produces cAMP. Subunits of G. proteins and others activated by phosphorylated enzyme-linked receptors can activate adenylate cyclase. In contrast, it is inhibited by inhibitory G. proteins. cAMP also activates other enzymes that further influence the expression of certain genes in the cell (Lynn).

IP3 and DAG derive from the PIP2 molecule phosphatidylinositor 4,5- bisphosphate by the enzyme phospholipase C (Lynn 2004). The enzyme is again stimulated by G. proteins and other proteins activated by phosphorylated enzyme-linked receptors. IP3 releases calcium from intracellular stores and, with DAG, can proceed to activate other enzymes, which in turn activate proteins that modify cell activity (Lynn).

Calcium can activate enzymes, such as protein kinase C. with DAG or bind to the calmodulin molecule, which in turn, can activate many other proteins, thus, produce a ripple effect and diverse cell activity and function (Lynn).

Cell communication, thus, happens through chemical signals and cellular receptors when there is direct contact of molecules between the surfaces of two cells or when a chemical signal is released and recognized by another proximate or distant cell (Department of Biology 2003). The circulatory systems take hormones to many locations and growth factors are released and act on proximate tissues. Ligands, on the other hand, are signals that bind cell surface receptors, such as insulin or pass into the cell and bind an internal receptor, such as steroid hormones (Department of Biology). Signal transduction is that cell activity to change in response to a receptor-ligand interaction, wherein the ligand is the primary messenger. With the binding, other molecules or second messengers develop within the target cell. These second messengers transmit the signal from one place to another, such as from the plasma membrane to the nucleus, and this series of changes occurs in the cell that can modify the cell's function or identity (Department of Biology). Messenger molecules may be amino acids, peptides, proteins, fatty acids, lipids, nucleosides or nucleotides. Hydrophobic messengers bind to intracellular receptors that regulate or influence expression of specific genes.

A ligand binds its receptor through specific weak non-covalent bonds by suiting into a specific binding site or "pocket." Binding of most of cognate receptors where a ligand has low concentrations means a high receptor affinity and low receptor affinity happens when a high concentration of the ligand is needed (Department of Biology). Prolonged exposure to a ligant, and the resulting occupancy of the receptor, often results in desensitization. Desensitization depends on receptor down-regulation through the removal of the receptor from the surface or by changing to the receptor that will lower the affinity to the ligand or disable it in initiating changes in cellular function (Department of Biology). Desensitization may likewise develop tolerance, which reduces or eliminates the effectiveness of some medicines when over prescribed.

A proteins are guanine-nucletide-binding that change the target protein's activity (Department of Biology 2003). Their receptors possess an extra-cellular N-terminus and a cytosolic C-terminus, which are separated by seven trans-membrane alpha helices, in turn, connected by peptide loops. The cytosolic loop between the 5th and 6th alpha helices binds a specific G. protein (Department of Biology).

A proteins that are bound to GTP are active, while those bound to GDP are not (Department of Biology 2003). These proteins are classified into large heterotrimeric and small monomeric G. proteins. When a messenger binds the heterotrimeric G. protein-linked receptor, it changes shape to allow linking with the trimeric G. protein (Department of Biology).

Many G. proteins make signal transduction events a diversified affair. Some bind potassium or calcium ion channels in neurotransmitters, while some either release or form major second messengers, such as cyclic AMP and calcium ions (Department of Biology 2003). Still some activate kinases. cAMP os produced by adenylyl cyclase, embedded in the plasma membrane and activated by binding an activated alpha subunit of the Gs G. protein. Phosphodiesterase progressively decreases cAMP in the absence of the ligand and active G. protein, thus reducing cAMP levels. Protein kinase A is a cAMP-dependent kinase, which is cAMP's main intracellular target (Department of Biology).

A protein signaling is quite important because its disturbance or disruption leads to several human diseases (Department of Biology 2003). For example, the micro-organism vibrio cholerae secretes the cholera toxin, which modifies the salt and fluid balance in the intestines. Hormones that activate Gs G. protein to increase cAMP normally maintain that balance. The disease occurs when the cholera toxin enzymatically changes Gs to disable it from converting GTP to GDP. Under this condition, Gs cannot be inactivated, keeping cAMP levels high and leading intestinal cells to secrete salt and water (Department of Biology). Extreme dehydration can lead to death.

The release of calcium ions is another key element in many signaling processes. Calcium ions help regulate many cellular functions (Department of Biology). Calcium ionophore releases calcium from the intracellular stores that imitate the effect of Insp3 activation. These stores can be released by the InsP3 channel and the ryanodine receptor channel, which opens when calcium is present. And although other proteins bind calcium to regulate its activity, binding to the protein calmodulin to produce a calcium-calmodulin complex is most frequently resorted to as an intermediate measure (Department of Biology).

The fertilization of animal ova illustrates calcium-mediated signal transduction, following a receptor-ligand interaction (Department of Biology 2003). The sperm, at first, binds the egg's surface at the membrane. Within 30 seconds, a wave of calcium releases spreads from the location of the sperm contact. This release of calcium determines two events. The first is the stimulation of the fusion of the cortical granules with the egg's plasma membrane to change or modify the surrounding coat of the egg and help prevent the binding of another sperm to it (Department of Biology). This is the natural and slow block to the phenomenon called polyspermy. The second event consists of the egg activation and the resumption of metabolic processes.

Nitric oxide is also a signal. It is a toxic and short-living gas molecule that plays a part as a signaling molecule in the cardiovascular system (Department of Biology 2003). It couples G. protein-linked receptor stimulation in endothelial cells to the relaxation of smooth muscle cells in blood vessels. This relaxation of the vascular smooth muscles or vasodilator involves six events or stages. First is the binding of acetylcholine to G. protein receptors that produces InsP3. Second is the release of calcium ions by InsP3 from endoplasmic reticulum. Third is the formation of complex calcium ions and calmodulin, which stimulates nitric oxide synthase in order to produce nitric oxide. Fourth is the diffusion of nitric oxide from the endothelial cell into adjacent smooth muscle cells. Fifth the activation of guanylyl cyclase in smooth muscle cells in order to produce cyclic GMP or cGMP. The sixth and last is the activation of the protein kinase G. To produce several muscle proteins that induce muscle relaxation (Department of Biology).

A ligand that binds a protein kinase-associated receptor stimulates a kinase activity and the blend of phosphorylation transmits the signal (Department of Biology 2003). Receptor tyrosine kinases assemble and go through autophosphorylation and begin a chain reaction, leading to cell growth, proliferation or differentiation. Most receptor tyrosine kinases have one trans membrane or TM domain, an extra-cellular ligand-binding domain and a cytosolic tail with tyrosine residue targets of the tyrosine activity.

Ligand binding begins the activation of the receptor tyrosine kinases, conducing to receptor aggregation, often as dimers. As soon as the epidermal growth factor receptor is autophosphorylated in response to the EGF ligand, a variety of GRB2 SH2-domain- containing and Sos guanine-nucleotide release protein binds the receptor. This change activates the Sos and causes the release of GDP, which in turn, allows Ras to bind a new GTP and get activated (Department of Biology). When activated, Ras creates a pathway, which includes the mitoen-activated protein kinases or MAPKs. These receptor tyrosine kinases stimulate a divergence of signaling pathways. Receptor tyrosine kinases can activate a form of phophlipase C. And phosphatidylinositor-3-kinase or PI3K (Department of Biology).

Growth factors act as messengers (Department of Biology 2003). Besides nutrients, growth factors are needed by the cell for growth. These include platelet-derived growth factors, insulin, insulin-like growth factor 1 or IGF-1, fibroblast growth factor or EGF, and nerve growth factor or NGF. This ligand function has a more significant value than growth and cell division. Impairment of the growth factor by affecting receptor tyrosine kinases can have telling consequences on embryonic development (Department of Biology). Fibroblast growth factors and fibrolast growth factor receptors have strong roles in both embryonic and adult signaling. These receptors play an important role in the development of the mesoderm, the embryonic tissue that eventually develops into muscle, cartilage, bone and blood cells (Department of Biology). When dimerized with normal versions of the receptor or becomes a mutant receptor, it will have a dominant inhibitory effect on normal activity as a dominant negative mutation. Such a mutant version of the receptor, when injected into frog eggs, can cause the failure of mesodermal tissue to develop. In the case of the frog experiment, tadpoles developed with heads but without bodies. Defects of this receptor in humans lead to achondroplasia or dwarfism and thanatophoric dysplasia or severe bone abnormalities, which are fatal in infancy (Department of Biology).

Hormones are chemical signals secreted by a tissue to regulate another, which is located at a distance (Department of Biology 2003), often transmitted through the circulatory system. They may be proteins, peptides, steroids or other molecules and their signals can be classified according to the distance they negotiate in reaching target cells.

Hormones can be proteins, peptides, steroids and other molecules. An endocrine hormone is transmitted by the circulatory system, while a paracrine hormone functions on proximate cells. Endocrine tissues directly secrete into the bloodstream while exocrine tissues do so into ducts, which bring the secretions to target parts of the body (Department of Biology). The pancreas secretes both endocrine and paracrine hormones. Insulin and glucagons are the endocrine hormones it secretes, while its paracrine hormones are digestive enzymes. Endocrine hormones eventually reach target tissues, such as the heart and liver, in the case of epinephrine, or liver and skeletal muscles, in the case of insulin (Department of Biology).

Endocrine hormones are categorized into four. These are amino acid derivatives or epinephrine; peptides, such as anti-diuretic hormone or vasopressin; proteins, such as insulin; and lipid-like hormones, including steroids such as testosterone.

Paracrine hormones, on the other hand, include a histamine, which is a histine derivative, and the prostaglandins, which arachidonic acid derivatives (Department of Biology).

Cells also regulate what is called programmed cell death or apoptosis, a well-ordered way of eliminating cells (Department of Biology 2003). Apoptosis is, however, quite different from necrosis, which is cellular death from massive tissue injury or destruction. Apostosis is a normal function of the living organism, such as the removal of the webbing of fingers and toes in embryos, extra neurons in infants and old blood cells in adults (Department of Biology). This condition activates specific proteases called caspases from the procaspases. Fas ligands on the surface of lymphocytes bind the Fas receptors on the infected cell surface. This leads to the clustering of the Fas ligands, the attaching of adapter proteins and the gathering of procaspases at the location or site. The procaspases activate one another to produce a series of conditions that ends in apoptosis (Department of Biology).

CONCLUSION

Medical practitioners and researchers have greatly benefited from the review made and published by Dr. Gilman on signal propagation through heterotrimeric G. protein. A more thorough discussion or appreciation of the physiological regulation needs a study of the members of the families and super-families of the basic elements of the system, such as GPLR, heterotrimeric G. proteins and their effectors (Morris and Malbon 1999). Each level or segment of these families has growth impressively. New partners of protein-protein interactions have been discovered since then in each level or segment. RAMP molecules, in addition to scaffolding cytoskeletal and adaptor molecules were discovered only a few years ago. In the meantime, RGS molecules, their target G. proteins and their biochemical and physiological modes of regulation are current focuses of research. Effectors for G. proteins now also include tyrosine kinases, like Btk, while both G. proteins and GPLR have emerged as targets for tyrosine kinases. G proteins retain their strong rles in more complex biological processes, such as oncogenesis, differentiation, development and insulin action (Morris and Malbon). Disturbance of these complex pathways by mutation has been known to develop into conditions like the McCune Albright Syndrome. In the meantime, a better appreciation of human pathologies requires a broader treatment with the necessary physiological, chemical and molecular features of signaling and alterations to it.