Structure and Organization of Membranes

The first living cell probably came into being when a membrane formed, separating that cells precious contents from the rest of the universe. Membranes define the external boundary of cells and regulate the molecular traffic across that boundary. Membranes are tough but flexible, self-sealing, and selectively permeable to polar solutes. Their flexibility permits the shape changes that accompany cell growth and movement (such as amoeboid movement). Their ability to seal over temporary brea s in their continuity allows two membranes to fuse, as in exocytosis, or a single membrane-enclosed compartment to undergo fission, yielding two sealed compartments, as in endocytosis or cell division, without creating gross lea s through the cell surface. !ecause membranes are selectively permeable, they retain certain compounds and ions within cells and within specific cellular compartments, and exclude others. "ll biological membranes, whether from eu aryotic or pro aryotic cells, have the same classes of chemical components, a similarity in structural organi#ation, and a number of properties in common. There are ma$or differences in the specific lipid, protein, and carbohydrate components but not in the physicochemical interaction of these molecules in the membrane. Membranes are not merely passive barriers. They include an array of proteins speciali#ed for promoting or cataly#ing a variety of molecular events. %umps move specific organic solutes and inorganic ions across the membrane against a concentration gradient& energy transducers convert one form of energy into another& receptors on the plasma membrane sense extracellular signals, converting them into molecular changes within the cell. 'ellular membranes control the composition of the space they enclose not only by their ability to exclude a variety of molecules but also because of the presence of selective transport systems permitting the movement of specific molecules from one specific molecules from one side to the other. !y controlling the translocation of substrates, cofactors, ions, and so on, from one compartment to another, membranes modulate the concentration of substances, thereby exerting an influence on metabolic pathways. The plasma membrane of eu aryotic cells also has a role in cell-cell recognition, maintenance of the shape of the cell, and in cell locomotion. The site of action of many hormone and metabolic regulators is on the plasma membrane, where there are specific receptors, and in the information to be imparted to the cell by the hormone or regulator is transmitted by the

membrane component to the appropriate metabolic pathway by a series of intracellular intermediates, termed second messengers. Typical Structure of Biological Membranes Typically, a biological membrane contains lipid, protein, and carbohydrate in ratios varying with the source of the membrane. The carbohydrate is covalently associated with protein (glycoproteins) or with lipid (glycolipids and lipopolysaccharides). Thus the membrane can be thought of as a lipid-protein matrix in which specific functions are carried out by proteins, while the permeability barrier and the structural integrity of the membrane are provided by lipids. The one membrane structure common to all cells is the plasma membrane. This membrane encapsulates the cytoplasm and creates internal compartments in which essential functions are carried out. (n addition to its role as a physical barrier that maintains the integrity of the cell, the plasma membrane provides functions necessary for the survival of a cell, including exclusion of harmful substances, acquisition of nutrients and energy sources, disposal of unusable and toxic materials, reproduction, locomotion, and interaction ith components in the environment . "ll these functions re)uire coordination both for short-range processes, such as sensation, and for long-range processes, such as growth and differentiation. "lthough some characteristics of biological membranes can be explained by the properties of membrane lipids in a)ueous solution, other characteristics, especially the ability to perform function such as transport and en#ymatic activities, depend on the presence of membrane-associated proteins. The Molecular !onstituents of Membranes *ne approach to understanding membrane function is to study membrane composition-to determine, for example, which components are commonly present in membranes and which are unique to membranes with specific functions. %roteins and polar lipids account for almost all of the mass of biological membranes& the small amount of carbohydrate present is generally part of glycoproteins or glycolipids. The relative proportions of protein and lipid differ in different membranes, reflecting the diversity of biological roles. The myelin sheath, which serves as a passive electrical insulator wrapped around certain neurons, consists primarily of lipids, but the membranes of

bacteria, mitochondria, and chlorophasts, in which many en#yme-cataly#ed metabolic processes ta e place, contain more protein than lipid. The "ipid !onstituents of Biological Membranes "ll biological membranes contain lipids as ma$or constituents. The molecule that play the dominant roles in membrane formation all have highly polar head groups and, in most cases, t o hydrocarbons tails. This composition ma es molecular sense as if a large head group is attached to a single hydrocarbon chain, the molecule is wedge-shaped and will tend to form spherical micelles. " double tail yields a roughly cylindrical molecule& such cylindrical molecules can easily pac in parallel to form extended sheets of bilayer membranes with the hydrophilic head groups facing outward into the a)ueous regions on either side. The four ma#or classes of membraneforming lipids $ glycerophospholipids, sphingolipids, glycosphingolipids, and glycoglycerolipids $ share this type of cylindrical molecular structure% They differ principally in the nature of the head group%

,ig-. %hospholipids and membrane structure.

&lycerophospholipids +lycerophospholipids (also called phosphoglycerides) are the ma$or class of naturally occurring phospholipids, lipids with phosphate-containing head groups (,ig-). These compounds ma e up a significant fraction of the membrane lipids throughout the bacterial, plant, and animal ingdoms. "ll glycerophospholipids can be considered to be derivatives of glycerol-.phosphate. 'arbon / in glycerol-.-phosphate is a chiral center, and the

naturally occurring glycerophospholipids are derivatives of the 0 enantiomer. The sterochemical configuration of the general structure of glycerophospholipids is shown in ,ig /a. ,ig/b. shows the molecule in the manner it will be generally used to represent membrane lipids, with the hydrophilic tails drawn to the right and the hydrophilic head group to the left.
,ig/3 +lycerophospholipid structure

1sually 2- and 2/ are acyl side chains derived from the fatty acids& often one is saturated, the other unsaturated. The hydrophilic 2 . group varies greatly, and it is this that confers the greatest variation in properties among the glycerophospholipids (,ig..)
,ig.. The hydrophilic groups (2. in ,ig / that distinguish common glycerophospholipids). (n addition to this variation, there is also variation in the hydrocarbon tails (2-, 2/) in the structures shown in ,ig.).

Table'( "ipid composition of some biological membranes

The simplest members of the group, phosphatidic acid, is only a minor membrane constituent& its principal role is as an intermediate in the synthesis of other glycerophospholipids. The names of glycerophospholipids are derived from phosphatidic acid 3 phosphatidylcholine, phosphatidylethanolamine, and so on. "s ,ig. shows, the glycerophospholipids have polar head groups, all carrying some charge. !ecause the hydrocarbon tails are derived from the naturally occurring fatty acids in various combinations, an enormous variety of glycerophospholipids exists. ,or example, the erythrocyte membrane contains molecules with hydrocarbon chains of -4 to /5 carbons, with 6 to 4 double bonds. 7uch variation in membrane composition allows 8fine-tuning9 of membrane properties for the diverse functions that different membranes must perform. Spingolipids and &lycosphingolipids " second ma$or class of membrane constituents is built on the long-chain amino alcohol sphingosine, rather than on glycerol. (f a fatty acid is lin ed via an amide bond to the :;< / group, the class of sphingolipids referred to as ceramides is obtained. 'eramides consist only of sphingosine and a fatty acid. ,urther modification, by addition of groups to the '-- hydroxyl of sphingosine, leads to a variety of other sphingolipids. "n essentially

important example is sphingomyelin, in which a phosphocholine group is attached to the '-. hydroxyl.

(n some of the membrane lipids built on sphingosine, the head group contains saccharides. 0ipids containing saccharide groups go under the general name of glycolipids. The glycosphingolipids constitute the third ma$or class of membrane lipids. They include such molecules as the cerebrosides (monoglycosyl ceramides) and gangliosides, anionic glycosphingolipids containing one or more sialic acid residues. "s the names of the compounds suggest, they are especially common in the membranes of brain and nerve cells. &lycoglycerolipids "nother class of lipids, less common in animal membranes but widespread in plant and bacterial membranes, are the glycoglycerolipids, exemplified by monogalactosyl diglyceride. This compound may actually be the most abundant of all polar lipids, for it constitutes about half the lipid in chloroplast membranes. 7uch lipids are also abundant in archaebacteria, where they are the ma$or membrane component. !holesterol *ne important lipid constituent of many membranes bears little superficial resemblance to the compounds. This substance is cholesterol, which is a member of a large group of substances called steroids. 7teroids include a number of important hormones, among them the sex hormones of higher animals. (n fact, cholesterol is the precursor for the synthesis of many of these substances. 'holesterol is a wea amphipathic substance, because of the hydroxyl group at one end of molecule. <owever, some of the cholesterol present in membranes has been rendered more hydrophobic by esterification of the

hydroxyl group to a fatty acid. "s the conformational structure in ,ig===was the fused cyclohexane rings in cholesterol are all in the chair conformation. This ma es cholesterol a bul y, rigid structure as compared with other hydrophobic membrane components such as the fatty acid tails. The cholesterol molecule fits aw wardly into membrane lipids and tends to disrupts regularity in membrane structure. This property can have a ma$or effect, because cholesterol constitutes />? or more of the lipid content in some membranes. 'hanges in membrane regularity can have profound effects on such properties as membrane stiffness and permeability. The Structure and )roperties of Membranes and Membrane )roteins The membranes of living cells are remar able bits of molecular architecture, with many and varied functions. Much of our current understanding biological membranes is based upon the fluid mosaic model proposed by 7. @. 7inger and +.0. ;icholson in -AB/. The fluid, asymmetric lipid bilayers carries within it a host of proteins. 7ome of them, called peripheral membrane proteins, are exposed at only one membrane face of the other. They are held to the membrane by interaction with lipid heads or integral membrane proteins. The integral membrane proteins are largely buried within the membrane but are usually exposed on both faces. (ntegral proteins are fre)uently involved in transmitting either specific substances or chemical signals through the membrane. The whole membrane is a mosaic of lipids and proteins.

Membrane )roteins are *ntegral or )eripheral Membrane proteins are classified as peripheral or integral. %eripheral proteins are probably bound to the membrane as a result of specific interactions with exposed, hydrophilic portions of integral membrane proteins. "s a conse)uence they can be dissociated from isolated membranes by agents that disrupt ionic or hydrogen bonds, such as high salt, CDT" +which chelates divalent cations), or urea. (n contrast, integral membrane proteins appear to be deeply embedded in the membrane. They can be released from the membrane only by disrupting the hydrophobic interactions of membrane lipids with organic solvents or detergents. 7ignificant

hydrophobic interactions with membrane lipids and proteins probably are responsible for the interaction properties of integral proteins. Cven after integral proteins have been solubili#ed, removal of the detergent may cause the protein to precipitate as an insoluble aggregate. The insolubility of integral membrane proteins results from the presence of domains rich in hydrophobic amino acids& hydrophobic interactions between the protein and the lipids of the membrane account for the firm attachment of the protein.

Some *ntegral )roteins ,ave ,ydrophobic Transmembrane -nchors (ntegral membrane proteins generally have domains rich in hydrophobic amino acids. (n some proteins, there is a single hydrophobic se)uence in the middle of the protein (as in glycophorin) or at the amino or carboxyl terminus. *ther membrane proteins have multiple hydrophobic se)uences, each long enough to span the lipid bilayer when in the -helical conformation. <ydrophobic interactions between the nonpolar amino acids and the acyl side chains of the membrane lipids firmly anchor the protein in the membrane, providing a transmembrane pathway for proton translocation. ,rom the structures of integral membrane proteins that have been determined or inferred so far, a number of generali#ations can be drawn---. " significant proportion of the polypeptide must interact with membrane lipids through hydrophobic interactions. /. -helices are common intramembrane structures of polypeptides. .. The main carbohydrate portions of plasma membrane glycoproteins are always found on the outside of the cell.

5. <ydrophilic regions of membrane proteins are exposed at the membrane surface, although the si#es and locations of these domains (inside or outside) are highly variable among different membrane proteins.

<ydrophobic <ydrophilic

,ig -3 %lots of hydropathy index against residue number for four integral membrane proteins.

+iven these ground rules, many wor ers have tried to predict the intramembrane structure of membrane proteins from their primary amino acid se)uences alone, and they have had some success. ,or example, hydrophobic, membrane-spanning -helices can be predicted by loo ing for hydrophobic stretches of amino acids in the primary se)uence that are long enough to span the membrane (about /6 residues). 'omputer algorithms octanol program of .MBOSS softwares have been developed to identify such regions in membrane proteins& these algorithms yield hydropathy plots. The se)uences of most integral proteins contain one or more regions rich in hydrophobic residues and long enough to span the .nm thic lipid bilayer. "n -helical peptide of /6 residues is $ust long enough to span the bilayer (length per residue is 6.-> nm). !ecause a polypeptide chain surrounded by lipids has no water molecules with which to form hydrogen bonds, it will tend to fold into -helices or sheets, in which intrachain hydrogen bonding is maximi#ed.

7everal simple methods of analy#ing amino acid se)uences have been found to yield reasonably accurate predictions of secondary structure for transmembrane proteins. The relative polarity of each of the /6 amino acids has been determined experimentally by measuring the free-energy change of moving a given residue from a hydrophobic solvent into water. This free energy of transfer ranges from very exorgonic for changes or polar residues to very endergonic for amino acids with aromatic or aliphatic hydrocarbon side chains (Table -). To estimate the overall hydrophobicity of a se)uence of amino acids, one sums the free energies of transfer for those residues, obtaining a hydropathy index for that region. To search a se)uence for potential membrane E spanning segments, one calculates the hydropathy index for successive segments of a given si#e (a 8window9, which may be from B to /6 residues). ,or a window of B residues, the indexes for residues - to B, / to F, . to A, and so on, are plotted as in ,igure -. " region of /6 residues of high hydropathy index is presumed to be a transmembrane segment. Ghen the se)uences of membrane proteins of nown three-dimensional structure are scanned in this way, a reasonably good correspondence is found between predicted and nown membranespanning segments. <ydropathy analysis predicts a single hydrophobic helix for glycophorin (,ig -a), five for the M sub-unit of the photosynthetic reaction center protein (,ig -b), seven transmembrane segments for bacteriorhodopsin (,ig -c), and t elve segments for the chloride-bicarbonate exchanger (,ig -d). *ctanol3 -n .MBOSS program +computer-based algorithm/ used to display protein hydropathy% .MBOSS +.uropean Molecular Biology Open Soft are Suite/ is an open source pac0age of sequence analysis tools% This soft are covers a ide range of functionability and can handle in a variety of formats%

1escription
%rotein se)uences that form transmembrane regions are assumed to have a thermodynamic preference for a hydrophobic environment (inside the membrane lipid bilayer), rather than an a)ueous environment in water. The free energy change for each amino acid residue between a lipid and a water environment can be measured experimentally, and the values for peptides can be shown to be additive (Ghite and Gimley -AAA). The octanol program calculates two free energy differences.

The first is the free energy difference between solution in water and association with the interface (glycerol group) of a %*%' (palmitoyloleoylphosphocholine) bilayer. The second is the free energy difference between water and octanol, e)uivalent to the environment inside a lipid bilayer. 2esidues which can be buried inside a lipid bilayer must be in a region of the peptide where most residues show a free energy difference in favour of being in an octanol environment or at least being in the lipidHwater interface region. Ghite and Gimley (-AAA) showed that a sliding window of either free energy difference will indicate the location of probably transmembrane regions, but that the best indicator is the difference between the two values, which is the free energy difference between the interface and octanol environments. The free energies are calculated over a sliding window of -A residues, about the si#e of a membrane spanning alphahelix. The energy values for each residue are added over the window.

1atabase entry( ts (opsd2human

ID OPSD_HUMAN STANDARD; PRT; 348 AA. AC P08100; Q16414; DT 01-AUG-1988 (Rel. 08, C e!"e#$ DT 01-AUG-1988 (Rel. 08, %!&" &e'(e)*e (+#!"e$ DT 1,--U%-1999 (Rel. 38, %!&" !))."!"/.) (+#!"e$ D0 RHODOPSIN. GN RHO. OS H.1. &!+/e)& (H(1!)$. OC 0(2! 3."!; Me"!4.!; C5. #!"!; C !)/!"!; 6e "e7 !"!; M!11!l/!; OC 0("5e /!; P /1!"e&; C!"! 5/)/; H.1/)/#!e; H.1.. RN 819 RP S0QU0NC0 :ROM N.A. R; M0D%IN0; 84<=<=<9. RA NATHANS -., HOGN0SS D.S.; RT >I&.l!"/.) !)# )(*le."/#e &e'(e)*e .? "5e @e)e e)*.#/)@ 5(1!) RT 5.#.+&/).>; R% P .*. N!"l. A*!#. S*/. U.S.A. 81A48,1-48,,(1984$. RN 8<9 RP S0QU0NC0 O: 1-1<0 :ROM N.A. RA B0NN0TT -., B0%%0R B., SUN D., CARICO C.; R% S(71/""e# (NO6-1994$ ". "5e 0MB%DGe)B!)2DDDB- #!"!7!&e&. RN 839 RP R06I0E ON ADRP 6ARIANTS. R; M0D%IN0; 9400490,. RA A%-MAGHTH0H M., GR0GORF C., ING%0H0ARN C., HARDCAST%0 A., RA BHATTACHARFA S.; RT >R5.#.+&/) 1("!"/.)& /) !(".&.1!l #.1/)!)" e"/)/"/&

+/@1e)".&!.>; R% H(1. M("!". <A<49-<,,(1993$. RN 849 RP 6ARIANT ADRP HIS-<3. R; M0D%IN0; 901369<<. RA DRF-A T.P., MCG00 T.%., R0ICH0I 0., HAHN %.B., COE%0F G.S., RA FAND0%% D.E., SANDB0RG M.A., B0RSON 0.%.; RT >A +./)" 1("!"/.) .? "5e 5.#.+&/) @e)e /) .)e ?. 1 .? e"/)/"/& RT +/@1e)".&!.>; R% N!"( e 343A364-366(1990$. RN 8,9 RP 6ARIANTS ADRP. R; M0D%IN0; 910,1,=4. RA :ARRAR G.-., C0NNA P., R0DMOND R., MCEI%%IAM P., BRAD%0F D.G., RA HUMPHRI0S M.M., SHARP 0.M., ING%0H0ARN C.:., BASHIR R., -AF M., RA EATTF A., %UDEIG M., SCHING0% A., SAMANNS C., GA% A., RA BHATTACHARFA S.S., HUMPHRI0S P.; RT >A(".&.1!l #.1/)!)" e"/)/"/& +/@1e)".&!A !7&e)*e .? "5e 5.#.+&/) RT + .l/)e--H5/&"/#/)e &(7&"/"("/.) (*.#.) <3$ /) +e#/@ ee& ? .1 RT 0( .+e.>; R% A1. -. H(1. Ge)e". 4=A941-94,(1990$. RN 869 RP 6ARIANTS ADRP HIS-<3; ARG-,8; %0U-34= AND S0R-34=. R; M0D%IN0; 9101,<=3. SQ PROT0IN S0QU0NC0 348 AA; 3889< ME; 0=443B0A CRC3<; MNGTEGPNFY VPFSNATGVV RSPFEYPQYY LAEPWQFSML AAYMFLLIVL VTVQHKKLRT PLNYILLNLA VADLFMVLGG FTSTLYTSLH GYFVFGPTGC GEIALWSLVV LAIERYVVVC KPMSNFRFGE NHAIMGVAFT WVMALACAAP EGLQCSCGID YYTLKPEVNN ESFVIYMFVV HFTIPMIIIF FCYGQLVFTV ATTQKAEKEV TRMVIIMVIA FLICWVPYAS VAFYIFTHQG SNFGPIFMTI YNPVIYIMMN KQFRNCMLTT ICCGKNPLGD DEASATVSKT ETSQVAPA

GFPINFLTLY NLEGFFATLG PLAGWSRYIP KEAAAQQQES PAFFAKSAAI

Output file format octanol draws a graph showing the free energy calcuated over a sliding window. I*ctanol resultJ
The line on the default plot is the difference between the interface and octanol free energy calculations. 'ommand line options allow the display of the interface and octanol values, or hiding the difference values. (n the example, the human opsin protein has B transmembrane regions3 .B-4-, B5AF, --5--.., ->.--B4, /6.-/.6, />.-/B4 and /F>-.6A. Cach is about /6 residues in length, which is also the gap between tic mar s on the se)uence axis. "ll have energetic preferences for being in the lipid (octanol) enviroment - shown as being above the #ero line - or have at least no clear preference.

2unning octanol with all three plots3 gives a graph with the water-interface and water-octanol plots. ,or those regions where the diference plot is close to #ero, both the other two plots are above the line, showing a preference for either the octanol or the interface membrane environments rather than water.

3eceptor-)rotein( Membrane receptors consist of transmembrane domains and the ligand domains, functional domains, which for many membrane receptors involve protein inase activities. (n additional, specific immunological domains contain primary epitopes of antigenic regions. -adrenergic receptor( 7everal membrane receptors have been cloned and studied with regard to structure and function, including the -receptors (- and /), which recogni#e catecholamines, principally norepinephrine, and stimulate adenylate cyclase. -- and /- receptors are subtypes that differ in affinities for synthetic anatagonists. Thus, --adrenergic receptor binds norepinephrine with a high affinity than epinephrine, whereas the order of affinities is reversed for the /-adrenergic receptor. The drug isoproterenol has a greater affinity for both receptors than the two hormones. (n ,ig /, the amino acid se)uence is shown (with single letter abbreviations for amino acids& for the /-adrenergic receptor. " polypeptide stretch extending from -helix ( extends to the extracellular space. There are seven membrane-spanning domains and these appear also in the - receptor where there is extensive homology with the / receptor. 'ytoplasmic peptide regions extend to form loops from ( to ((, ((( to (K, K to K( and an extended chain from K((. The long extended chain from K(( may contain sites of phosphorylation (serine residues) of the receptor, which is part of the receptor regulation process involving receptor desensiti#ation. 'ell exterior peptide loops extend from (( to (((, (K to K, and ta e part in ligand binding. (t appears that ligand binding may occur in a poc et arranged by the location of the membranes-spanning cylinders (-K((, ,ig .. 2ecently, reported wor suggests that the sixth transmembrane domain may play a role in the stimulation of the adenylate cyclase activity. !y substitution of a specific cysteine residue in the sixth transmembrane domain, a mutant was produced that displays normal ligand binding properties but a decreased ability to stimulate the cyclase.

,ig /3 %roposed model for the insertion of the / adrenergic receptor ("2) in the cell membrane. This model is based on hydropathy analysis of the human /"2. The standard one letter code for amino acid residues is used. <ydrophobic domains are represented as transmembrane helices.

Molecular mechanism arrangement of the adrenergic receptor Signal Transduction

4ig5()roposed

of

helices in the membrane% *t is also proposed that helices signal *6, 6*, and 6** !ellular transduction reside in the membrane is a tso as o-step process3 to delineate a ligand binding poc0et, ith helix 6** centrally located% -. ,irst, a signaling

molecule is sensed by a receptor at a target cell

and then the receptor is activated. Membrane-bound receptors respond to a large spectrum of extracellular signals. Cxternal signals range from light and odours to hormones, growth factors, and cyto ines. /. Ghen the receptor sensing the signal is a catalyst, a inase, the response is amplified. "s diversified as the signals, are the proteins, which respond to them. (n each case, binding of a signaling molecule converts the dormant receptor to an activate state. " mechanism involved in the transition of a receptor from its inactive to its active state is receptor oligomeri#ation. "ll receptors that transmit signals from the surface of the cell to the interior, and finally to the nucleus and the genes, have two features in common3 (-) The signaling molecule binds to the extracellular domain of the membrane-inserted receptor& and (/) 0igand binding triggers, in a cooperative manner, a change in the domain inside the cell. 1pon binding the ligand and followed by activation, ligand-receptor complexes are eventually internali#ed. (nternali#ed ligand-receptor complexes are dissociated in acidic endocyclic vesicles and the ligand is degraded in lysosomes, whereas the receptor may be degraded or recycled bac to the cell surface. 2eceptor-ligand complexes may be internali#ed together with proteins, which regulate their endocytosis and degradation. 2eceptor desensiti#ation by removal from the membrane and endocytosis is a feature shared by single-pass tyrosine inase receptors, and serpentine, heptahelical +-protein-coupled receptors. )hosphorylation " process, which, in most cases, modulates receptor signaling is phosphorylation. (n the case of +-protein-coupled heptahelical receptors, the interaction with specific inases is the first step in shutting off their action. (n other cases, binding of a growth factor to a receptor triggers the intrinsic receptor inase activity and leads to autophosphorylation. The important point is that the phosphates introduced in the receptor are essential for recognition and binding of other proteins, adaptors and transducers, which are often cytosolic protein inases and phosphatases. 7ignaling triggered by growth factor-receptor interactions leads to a response, which is often of global nature, such as growth, proliferation, and differentiation of cells. +rowth factors affect the cell cycle and the cell death programmes, which determine the fate of the cell. "lthough, many processes vital for the cell are affected, the main target is the genome. The essence of cellular

signaling is the transmission of signals from the surface of the cell to the nucleus and the subse)uent expression of genes. Dysfunction of the regulatory mechanisms controlling these processes can cause malignant transformation of cells and other diseases. 3eceptors for .pinephrine Trigger !yclic -M) )roduction Sutherland7s Model( The current understanding of the mechanism of epinephrine (and glucagons) action originated in the wor of Carl G. 7utherland, @r., and his colleagues in the early -A>6s. These investigators showed that epinephrine stimulates the activity of glycogen phosphorylase, which promotes the brea down of glycogen to glucose---phosphate, the rate-limiting step in the conversion of glycogen to glucose. 7utherlands laboratory identified adenosine .,>cyclic monophosphate (cyclic "M% or c"M%) as the intracellular messenger produced in response to extracellular epinephrine. ,ig 5 schemati#es the multistep path from the initial stimulus to the elevation of blood glucose. 7everal of these steps amplify the effect of hormone binding to the receptor, so that a single molecule of hormone can change the catalytic activity of thousands of en#yme molecules. Cventually, five proteins essential to the epinephrine response were identified and purified (,ig>)3 (-) a hormone receptor in the plasma membrane& (/) the en#yme adenylate cyclase, which cataly#es c"M% formation& (.) +s protein, which shuttles between the receptor and adenylate cyclase, activating the cyclase when hormone is bound to the receptor& (5) a c"M%-dependent protein inase, which phosphorylates target en#ymes within the cell, altering their activities& and (>) cyclic nucleotide phosphodiesterase, which degrades c"M% and thereby terminates the intracellular signal.

,ig 53 Cpinephrine triggers a series of reaction in hepatocytes in which catalysts activate catalysts, resulting in greater amplification of the signal. !inding of a small number of molecules of epinephrine to specific receptors on the cell surface activates adenylate cyclase. ,or convenient, 56 molecules of c"M% are produced by each molecule of adenylate cyclase. These 56 c"M% molecules activate -6 molecules of the protein inase, each of which in turn activates -6 molecule of the next en#yme in the cascade. The amplifications shown here for each step are probably gross underestimates.

(-)

,ig >3 The mechanism that couple binding of epinephrine (C) to its receptor (2ec) with the activation of adenylate cyclase molecule in the plasma membrane may be regulated by a stimulatory + protein, +s as shown or an inhibitory + protein, +i (not shown). +s and +i are under the influence of different hormones. <ormones that induce +T% binding to +i cause inhibition of adenylate cyclase, resulting in lower cellular levels of c"M%.

The .pinephrine- --drenergic 3eceptor !omplex

The action of epinephrine begins with the binding of the hormone to a protein receptor in the plasma membrane of a hormone-sensitive cell, a hepatocyte or myocyte, step (-). The binding is tight but noncovalent, li e the binding of an allosteric effector to an allosterically regulated en#yme. The binding site on the receptor is stereospecific and will accommodate only the natural hormone ligand or molecules with a closely similar threedimensional geometry. 7tructural analogs that bind to a receptor and mimic the effects of its natural ligand are called agonists& antagonists are analogs that bind without triggering the normal effect, and thereby bloc the effects of agonists. -adrenergic receptors are integral membrane proteins with amino acid se)uences that contain seven hydrophobic regions of /6 to /F residues, suggesting that the protein traverses the lipid bilayer seven times. The binding site for epinephrine is on the outer face of the plasma membrane& the hormone causes an intracellular change without itself crossing the plasma membrane. The binding of epinephrine apparently promotes a conformational change in the receptor, including the receptor domain that protrudes on the cytosolic face of the membrane. The first stage of hormone action of an allosteric effector on an allosterically regulated en#yme. The structural changes in the intracellular domain of the receptor allows its interaction with the second protein in the signal transduction pathway, a +T%-binding protein. &T)-Binding )rotein and -denylate !yclase (n the signal-transduction pathway, the next element is a protein called a stimulatory + protein, or +s, located on the cytosolic face of the plasma membrane. (+s ta es its name from the fact that, when bound to +T%, it stimulates the production of c"M% by adenylate cyclase, and en#yme of the plasma membrane). +s is composed of three polypeptides, , , and . (t is one of a large family of guanosine nucleotide-binding proteins that mediate a wide variety of signal transductions, including those triggered by many other hormones as well as certain sensory stimuli. +s can exist in either of two forms. Ghen its nucleotide-binding site (on the subunit) is occupied by +T%, +s is active and can interact with and activate adenylate cyclase. Gith +D% bound to the site, + s is inactive and incapable of activating adenylate cyclase. !inding of epinephrine cause the receptor to cataly#e the displacement of the +D% bound to inactive + s by

+T%& this converts +s to its active form, step (/). "s this occurs, the and subunits dissociate from the subunit& +s with adenylate cyclase converts the cyclase to its catalytically active form& the en#yme catalu#es the production of c"M% from "T%, raising the cytosolic level of this second messenger.

"ctivation of adenylate cyclase by +s is self-limiting& +s has a wea +T%ase activity and turns itself off by converting its bound +T% to +D% (,ig 4). The now inactive +s dissociates from adenylate cyclase, thereby inactivating it. "fter +s reassociate with the and subunits, +s again becomes available for interaction with hormone-bound receptor. 7ignal transduction through adenylate cyclase involves two steps in se)uence that amplify the original hormonal signal. ,irst, one hormone molecule bound to one receptor catalytically activates several + s molecules. 7econd, by activating a molecule of adenylate cyclase, one active +s molecule leads to the catalytic synthesis of many molecules of c"M%. The net effect of this cascade is a very significant amplification of the hormonal signal, which accounts for the very low concentration of epinephrine (and of other hormones) re)uired for activity. 'yclic "M%, the intracellular second messenger in this system, is shortlived, it is )uic ly degraded by cyclic nucleotide phosphodiesterase to >"M%, step (B), which is not active as a second messenger. The intracellular signal therefore persists only as long as the hormone receptor remains occupied by epinephrine. Methyl xanthines such as theophylline (a component of tea) inhibit the phosphodiesterase, potentiating the action of agents that act through adenylate cyclase.

,ig 43 The protein +s acts a selfinactivating switch.