MEMBRANE-MEDIATED COMMUNICATION

1.         Cells in a multicellular organism need to communicate with one another to:

a.         regulate their development into tissues.

b.         control their growth and division.

c.            coordinate their functions.

2.         They can communicate through intercellular junctions or display membrane-bound signaling molecules that influence other cells in direct physical contact.

3.         Types of Signaling:

a.            endocrine: hormones are carried in the blood to target cells throughout the body.

b.            paracrine: chemical mediators are rapidly metabolized so that they act on local cells only.

c.            synaptic: neurotransmitters act only on adjacent nerve cells through synapses.

d.            autocrine: paracrine signals act on the same cell type that produce the messenger hormone.

e.            juxtacrine:

i.          some cells express multiple repeats of growth factors such as transforming growth factor alpha (TGF) extracellularly on transmembrane proteins that provide an anchor to the cell.

ii.          this link to another cell with a TGF receptor.

iii.            important in producing local foci of growth in tissues.

4.         The chemical messengers include amines, amino acids, steroids, polypeptides.

5.         Types of Signaling molecules:

a.         small hydrophobic signaling molecules, eg. steroid and thyroid, diffuse through the plasma membrane of the target cell and activate receptor proteins inside the cell.

b.            hydrophilic signaling molecules, eg. neurotransmitters, paracrine signals, activate receptor proteins inside the cell.

c.            morphogens:

i.          these molecules determine the form of the body during development.

ii.          retinoic acid dictates the formation of limbs during embryonic life.

6.         In various parts of the body, the same chemical messenger can function as a neurotransmitter, a paracrine mediator, a hormone secreted by neurons into the blood and  a hormone secreted by gland cells into the blood.

7.            Receptors for Hormones, Neurotransmitters & other Ligands:

a.         The number of receptors for chemical messengers increase and decrease in response to various stimuli, and their properties change with changes in physiologic conditions.

b.         down regulation:

i.          when a signal molecule is present in excess, the number of active receptors decreases.

ii.            densensitization, in which receptors are chemically modified in ways that make them less responsive.

iii.         in receptor-mediated endocytosis, the receptors are taken into a transport vesicle together with the ligand which decreases the number of receptors in the membrane.

c.         up regulation: in the presence of a deficiency of the chemical messenger, there is an increase in the number of active receptors.

d.         these effects on receptors are important in explaining denervation and hypersensitivity.

e.         these receptors, which span the cell membrane, relay information to a series of intracellular intermediaries that pass the signal to its final destination in either the cytoplasm or the nucleus.

8.         Sites of action of chemical messengers:

a.         Cell membrane:

i.          some ligands bind directly to ion channels in the cell membrane, changing their conductance.

ii.          insulin increases uptake of glucose into the cell by altering the permeability of the membrane to glucose.

b.            Enzymes located in cell membrane:

i.            epinephrine and many peptide hormones bind to receptor sites on the cell membrane.

ii.          cause the release of a second messenger which initiates a sequence of enzyme mechanisms which produce the appropriate response.

iii.         some transmembrane catalytic receptors have an inherent enzymic activity as part of their structure.

iv.         in most cases, the enzyme activity is a tyrosine-specific protein kinase.

v.         several cell-surface receptors contain an extracellular domain for binding ligands and an intracellular domain with tyrosine kinase activity.

vi.         the binding of a ligand, such as insulin, to its receptor activates the intrinsic tyrosine kinase activity, which transfers the terminal phosphate group of ATP to the hydroxyl group of specific tyrosine residues of target proteins and of the receptor itself.

c.         Cellular organelles:

i.          one of the effects of thyroxin is seen at the level of the mitochondrion where it influences the enzymes of the electron carrier system involved in the formation of ATP.

ii.          much of the energy passing along the electron transport chain in those circumstances is lost as heat.

d.         Genes:

i.          steroid hormone pass through the cell membrane and bind to a receptor in the cytosol.

ii.          the complex formed passes to the cell nucleus where it exert a direct effect upon the chromosome by activating genes and stimulating transcription.

9.            Mechanism by which Chemical Messengers act:

a.         almost all the other ligands in the extracellular fluid bind to receptors on the surface of cells and many of them trigger the release of intracellular mediators such as cyclic AMP, IP₃ and DAG that initiate changes in cell function.

b.         hence, the extracellular ligands are called first messengers and the intracellular mediators are called second messengers.

c.         when activated, many of the membrane receptors initiate release of second messengers or other intracellular events via GTP-binding proteins.

d.         the second messengers generally activate protein kinases, enzymes that catalyze the phosphorylation of amino acids on proteins.

e.         addition of phosphate groups changes the configuration of the proteins, altering their functions and consequently the functions of the cell.

f.          in some instances, the intracellular portions of the receptors themselves are protein kinases.

g.         Table:

10.            Stimulation of Transcription:

a.         when thyroid and steroid hormones bind to their receptors inside cells, the conformation of the receptor protein is changed and a DNA-binding domain is exposed.

b.         the receptor-hormone complex moves to DNA, where it binds to enhancer elements in the untranslated 5’-flanking portions of certain genes.

c.         the binding of the receptor-hormone complex to DNA increases the transcription of mRNAs encoded by the gene to which it binds.

d.         the mRNAs are translated in the ribosomes, with the production of increased quantities of proteins that alter cell function.

e.         for glucocorticoid, estrogen, and progesterone receptors, the receptor is bound to a heat shock protein which covers the DNA-binding domain in the absence of the steroid.

f.          when the steroid binds to the receptor, the conformation change is release of the heat shock protein, exposing the DNA-binding domain.

g.         steroids can also bind to membrane receptors: rapid increase in Ca²⁺ concentration in sperm heads that is produced by progesterone and prompt steroid-induced alteration in the functions of various neurons.

11.            Structure of Steroid receptors:

a.         all these receptors have in common:

i.          a highly conserved cysteine-rich DNA-binding domain.

ii.          a ligand-binding domain at or near the C terminal of the receptor.

iii.         a poorly conserved N-terminal region.

b.         binding to the DNA occurs via zinc fingers.

12.       G Proteins:

a.         a common way to translate a signal to a biologic effect inside cells is by the way of nucleotide regulatory proteins or G proteins that bind GTP.

b.         in their resting state, G proteins, which consists of alpha, beta, and gamma subunits, are bound by the nucleotide GDP and have no contact with receptors.

c.         when a hormone or other first messenger binds to a receptor, the receptor causes the G protein to exchange GDP for GTP, which activates the G protein.

d.         the G protein then dissociates, after which the GTP-bound alpha subunit diffuses along the membrane and binds to an effector, activating it.

e.         after a few seconds, the alpha subunit converts GTP to GDP, thereby inactivating it.

f.          the alpha subunit will then reassociate with the beta-gamma complex.

g.         this family of G proteins are the larger heterotrimeric G proteins which couple

i.          cell surface receptors to catalytic units that catalyze the intracellular formation of second messengers.

ii.          the receptors directly to the ion channels.

h.         they are called serpentine receptors as they span the cell membrane 7 times.

13.            Intracellular Ca²⁺

a.         There exists an inwardly directed concentration gradient as well as an inwardly directed electrical gradient for Ca ions.

b.         much of the intracellular Ca²⁺ is bound by the endoplasmic reticulum and other organelles and these organelles provide a store from which Ca²⁺ can be mobilized to increased the concentration of free Ca²⁺ in the cytoplasm.

c.         Ca²⁺ enters cells through 2 kinds of channels:

i.            voltage-gated Ca²⁺ channels activated by depolarization.

ii.          ligand-gated Ca²⁺ channels activated by neurotransmitters and hormones.

iii.         in addition, there are Ca²⁺ channels that are activated by stretch.

d.         Ca²⁺ is pumped out of the cell

i.          in exchange for 2H⁺ by a Ca²⁺-H⁺ ATPase.

ii.          by an antiport driven by the Na⁺ gradient (3Na⁺ for each Ca²⁺).

e.         some second messengers act by increasing the cytoplasmic Ca²⁺ concentration.

f.          the increase in Ca²⁺ concentration is produced by:

i.            releasing Ca²⁺ from intracellular stores.

ii.            increasing the entry of Ca²⁺ into cells.

iii.         both mechanisms.

g.         the increase starts in one part of the cell and spreads to other parts of the cytoplasm.

h.         in many instances, the ligands initiate cyclic oscillations in cytoplasmic Ca²⁺ rather than a steady increase.

14.            Calcium Binding Proteins:

a.            troponin:

i.          Ca²⁺ binding protein involved in the contraction of skeletal muscle.

ii.          consists of 3 subunits: troponin I, troponin T and troponin C.

b.            calmodulin:

i.          the binding of four molecules of Ca²⁺ to calmodulin triggers a conformational change such that the activated Ca²⁺-calmodulin complex binds to and activates protein molecules.

ii.            calmodulin functions as an essential subunit of many complex proteins, including various calmodulin-dependent protein kinases, adenylate and guanylate cyclases, phosphodiesterase.

iii.         one of these is myosin light-chain kinase, which phosphorylates myosin.

iv.         this brings about contraction in smooth muscle.

v.         another is phosphorylase kinase, which activates phosphorylase.

vi.         Ca²⁺ / calmodulin kinases I and II are concerned with synaptic function.

vii.        Ca²⁺ / calmodulin kinase III is concerned with protein synthesis.

c.            calbindin

15.       Inositol Triphosphate and Diacylglycerol:

a.         the link between membrane binding of a ligand that acts via Ca²⁺ and the prompt increase in the cytoplasmic Ca²⁺ concentration is often inositol triphosphate (inositol 1,4,5-triphosphate, IP₃).

b.         when one of these ligands binds to its receptors, activation of the receptor produces activation of phospholipase C on the inner surface of the membrane via a G_q or G_11.

c.            phospholipase C catalyzes the hydrolysis of phosphophatidylinositol 4,5-diphosphate (PIP₂) to form IP₃ and diacylglycerol.

d.         the IP₃ diffuses to the endoplasmic reticulum where it triggers the release of Ca²⁺ into the cytoplasm.

e,         the Ca²⁺ released to the cytoplasm alters the activities of specific enzymes, either directly or by first binding to calmodulin.

f.          the diacylglycerol stays in the cell membrane where it activates one of the 7 subspecies of protein kinase C.

g.         protein kinase C requires Ca²⁺ for maximum affinity; diacylglycerol appears to act by increasing the affinity of protein kinase C for Ca²⁺ .

h.         the precursor of  PIP₂ is phosphatidylinositol which is found in small amounts in the inner lamella of the cell membrane.

I.          it is first converted to phosphatidyl 4-phosphate (PIP) and then to PIP_2.

j.          the IP₃ is metabolized by stepwise dephosphorylation to inositol.

k.            diacylglycerol is converted to phosphatidic acid and then to cytosine diphosphate (CDP) diacylglycerol which combines with inositol to form phosphodylinositol, completing the cycle.

l.            Synergism between messengers:

i.          the two second messengers, diacylglycerol and inositol 1,4,5-triphosphate, act synergistically to cause increased phosphorylation of proteins.

ii.            diacylglycerol activates protein kinase C by a process requiring Ca²⁺ whereas IP₃ elevates concentration of Ca²⁺ to activate calmodulin-dependent protein kinase.

iii.         the elevated levels of Ca²⁺ may act by additional mechanisms that do not involve calmodulin.

16.       Cyclic AMP

a.         also known as cyclic adenosine 3’,5’-monophosphate.

b.         formed from ATP by the action of the enzyme adenylate cyclase and converted to inactive 5’-AMP by the action of enzyme phosphodiesterase.

c.            phosphodiesterase itself is inhibited by methylxanthines such as caffeine and theophylline which augment the hormonal and transmitter effects mediated via cAMP.

d.         cyclic AMP activates protein kinase A which catalyzes the phosphorylation of proteins, changing their conformation and altering their activties.

e.            Components involved in change of cAMP concentration:

i.            adenylate cyclase: a catalytic unit that catalyzes the conversion of ATP to cAMP.

ii.            stimulatory and inhibitory receptors.

iii.            stimulatory and inhibitory G proteins that link the receptor to the catalytic unit.

f.            adenylate cyclase:

i.          a transmembrane protein that crosses the membrane 12 times.

ii.          eight isoforms have been cloned that interact with a variety of G proteins.

iii.         two bacterial toxins, cholera toxin and pertussis toxin have important effects on adenylate cyclase that are mediated by G proteins.

g.            Activation and Inactivation of adenylate cyclase:

i.          when the appropriate ligand binds to the inhibitory receptor, a G_i alpha subunit inhibits adenylate cyclase.

ii.          when the appropriate ligand binds to the stimulatory receptor, a G_s alpha subunit activates adenylate cyclase.

iii.         the energy for the activation and inactivation of adenylate cyclase is provided by GTP which the G protein hydrolyze to GDP.

iv.            heterotrimeric G proteins mediate the stimulatory and inhibitory effects on adenylate cyclase that are produced by many different ligands, yet the responses are specific.

v.         the specificity depends on the specificity of the receptor, which responds at low threshold to only one or a select group of related ligands.

h.         Ca²⁺ can interact with cAMP and the diacylglycerol-protein kinase C system interacts with the cyclic AMP-protein kinase A system.

i.          in some instances, the cAMP system inhibits the diacylglycerol system whereas in others, the diacylglycerol system facilitates the cyclic AMP system.

17.       Cyclic GMP and Guanylate Cyclase:

a.         cGMP is important in vision:

i.          light acts on rhodopsin in rods.

ii.            rhodopsin is linked to phosphodiesterase by G_t1

iii.            activation of phosphodiestrase accelerates the conversion of cGMP to 5’-GMP, terminating its actions.

b.            increases in intracellular cGMP activates cGMP-dependent kinase, producing physiologic effects.

c.            guanylate cyclases are a family of enzymes that catalyze the formation of cGMP.

d.         they are activated by nitric oxide and NO-containing compounds.

e.            membrane-bound guanylate cyclase differs from adenylate cyclase in that the enzyme is an integral part of the receptor.

f.          in contrast to cAMP, which affects a wide variety of processes, cGMP functions as a specialized messenger, being involved in smooth muscle relaxation, platelet aggregation and the visual system.

18.       Growth Factors:

a.         these are polypeptides and proteins.

b.         there are 3 groups:

i.          one group is made up of agents that foster the multiplication and development of various types of cells; nerve growth factor.

ii.          the lymphokines and cytokines are a second group produced by macrophages and lymphocytes and are important in the regulation of the immune system.

iii.         the third group is made up of colony-stimulating factors that regulate proliferation and maturation of red and white blood cells.

c.         many of the growth factors bind to receptors on the cell surface which have intracellular domains that are tyrosine kinases.

d.         when the ligand binds, the activated receptors trigger phosphorylation of tyrosine residues in the receptor itself and in neighboring proteins.

e.         this leads to changes in configuration of the proteins that alter cell function.

19.            Receptor Diseases:

a.            pseudohypoparathyroidism:

i.            parathyroid hormone fail to produce the increase in cAMP in their target organs.

ii.          one form appears due to a single-base substitution in the gene encoding the alpha subunit of G_s. 

iii.         this reduces the ability of parathyroid hormone to increase cAMP and produces its physiologic effects.

b.         a different activating mutation in G_s alpha is associated with the rough-bordered areas of skin pigmentation in the condition called McCune-Albright syndrome.

c.         in nephrogenic diabetes insipidus, vasopressin fail to produce increase in cAMP.

d.         certain diseases are caused by production of antibodies against receptors:

i.            antibodies against TSH receptors caused Grave’s disease.

ii.            antibodies against nicotinic acetylcholine receptors cause myasthenia gravis.