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MEMBRANE FUNCTION_ Signal Transduction

VIEWS: 20 PAGES: 9

  • pg 1
									                                                                                  Robert W. Baer, Ph.D
                                                                                         March 7, 1996


              MEMBRANE FUNCTION: Signal Transduction

General Goal: To describe how signaling molecules like neurotransmitters, hormones, and growth factors
initiate changes intracellular function by activating “second messenger” systems, ion channels, and
enzymes.
Specific Education Objectives: The student should be able to:
1. list the general types of receptor systems and differentiate between those most often used by
   neurotransmitters, hormones and growth factors.

2. draw an activation schematic of membrane G-protein system including the roles of receptor, G-protein
   subunits, effector and GTP.

3. describe the cAMP second messenger system and including the mechanism by which its production is
   increased, the mechanism by which it alters cell function, and the mechanism by which its action is
   terminated.

4. describe the IP3 and DAG second messenger system including the mechanism by which their
   production is increased, the mechanism by which each alters cells function, and the role played by G-
   proteins in its inactivation.

5. describe the cGMP second messenger systems including a differentiation between the roles of
   membrane bound guanylate cyclase and soluble guanylate cyclase.

6. describe the NO second messenger system including control of its production, its sphere of influence,
   and mechanism of action.

7. describe Ca++ as a second messenger including mechanisms by which intracellular concentrations are
   increased and the molecular form in which it exerts its action.

8. name the major types of cholinergic receptors and describe the signal transduction mechanisms
   associated with receptor binding.

9. name the major types of adrenergic receptors and describe the signal transduction mechanisms
   associated with receptor binding.

Resources:
       Lecture:        Dr. Baer
       Reading:        Ganong, W.F. Review of Medical Physiology (16th ed.). Appleton & Lange, 1993.
                       Chapter 1, pp. 30-40.
Page 2 Membrane Function: Signal Transduction


I.        INTRODUCTION TO RECEPTORS & SIGNAL TRANSDUCTION

          A.         Receptors are proteins associated with cells that recognizes neurotransmitters, hormones,
                     and drugs.

          B.         Signaling molecules transmit their information to cells in a variety of ways.

                                                               Transmitters
                                                                                     Transmitters
                                                                Hormones



                                                                                 
                                                                                

                                                             Second
                          Growth                           Messangers                 Ion
                                                 Tyrosine
                          Factors                                                     Channels
                                                 Kinase
                                                          Protein Kinases
                          Hormones:
                           Steroids                                     mRNA
                           Thyroid                                     Synthesis
                                                                   Protein Synthesis



                                                                 Figure 1


                     1.        Neurotransmitters bind with receptors that also act as ion channels or they
                               interact with G-proteins to stimulate effector enzymes to produce “second
                               messengers”.

                     2.        Most hormone receptors interact with G-proteins to alter “second messenger”
                               levels. Steriod hormone receptors are cytosolic receptors which travel to the
                               nucleus to alter mRNA synthesis.

                     3.        Most growth factor receptors have intrinsic tyrosine kinase activity and cause
                               phosphorylation of tyrosine residues on specific proteins.

          C.         Neurotransmitters tend to fall into one of several classes.

Table 1. Neurotransmitters know and putative.
                   BIOGENIC AMINES            PEPTIDES                AMINO ACIDS            OTHER
                  CATECHOLAMINES Substance P                          EXCITATORY             Nitric oxide (NO)
                  Epinephrine                 Neuropeptide Y          Glutamate              ATP
                  Norepinephrine              Enkephalins             Aspartate              Zinc
                  Dopamine                    Somatostatin                                   Arachidonic acid
                  INDOLAMINES                 VIP                     INHIBITORY             PAF
                  Serotonin (5-HT)                                    -Aminobutyric acid    Carbon monoxide
                  Histamine                                           Glycine
                  ESTERS
                  Acetycholine


          D.         The G-proteins involved in most signaling processes are trimeric proteins composed of ,
                      , and -subunits.
                                                                         Membrane Function: Signal Transduction Page 3


                                                              A




                                                                  R
                                                                                
                                                                           
                                                                                
                                            A                             GDP
                                                                                              A

                                     R                                                        R
                                                                                                     
                                                                                                 
                                                                                                     
                                            GTP
                                                                  A
                                                (GTPase)                                GTP               GDP
                                                    -Pi
                                                                  R
                                                                                    
                                                                           
                                                                                    
                                                                          GTP




                                                                      Adenylate Cyclase
                                                                       Phospholipase C
                                                                         Ion Channels
                                                                       Phospholipase A2
                                                                      Phosphodiesterase


Figure 2. G-Protein Cycling. G-protins are trimeric which cleave GTP (hence the name).. The alpha subunits (and probably the beta-
gamma subunit as well) exerts their effects when dissociate by the binding of GTP. Activity is terminated by intrinsic GTPase activity that
changes bound GTP into bound GDP. Symbols are: A = agonist; R = receptor; , , and  are the three subunits found in most membrane-
associated G-proteins.

                     1.        Occupation of the receptor protein causes it to associate with the G-protein.

                     2.        The receptor/G-protein complex allows a GTP to displace a GDP from the -
                               subunit.

                     3.        Binding of GTP causes dissociation into free receptor, free (GTP) -subunit, and
                               free   complex.

                     4.        The (GTP)- -subunit interacts with an effector protein to produce second
                               messenger or occasionally to open ion channels.

                     5.         -complexes were initially thought to be inert. This is probably not so, but their
                               exact role is various signaling processes remains to be fully defined.

                     6.        The -subunit has intrinsic GTPase activity so the attached GTP is turned into
                               GDP. This inactivates the -subunit and causes it to rebind the  -complex.

          E.         Binding of receptors to G-proteins can elicit the production or release of a large variety
                     of second messengers including: cAMP, inositol triphosphate (IP3), diacylglycerol
                     (DAG), cGMP, Ca++, and nitric oxide (NO). These agents in turn initiate other cellular
                     responses.

          F.         There are several families of G-proteins characterized by the unique structure of their -
                     subunits. The  and  subunits appear to be similar across families.
Page 4 Membrane Function: Signal Transduction


II.       THE cAMP SECOND MESSENGER SYSTEM

                                              As                                            Ai

                                                               Adenylate
                                             R1                 Cyclase                     R2
                                                  Gs                                   Gi


                                                    GTP                        GTP
                                           GDP                                              GDP
                                                                       ATP
                                   AMP
                                                PDE         cAMP


                                                                                                 C
                                                                                                     Protein
                                                    C
                                              Reg                                Reg
                                                    C                                            C


                                       Protein Kinase A                         PKA
                                            (PKA)                                            Protein-P

Figure 3. cAMP as a second messanger. cAMP is made from ATP by the enzyme adenylate yclase. CAMP is broken down by phosphodiesterase
(PDE). The activity of adenylate cyclase can be modulated in by G-proteins. When stimulatory agonixts (As)bind with their receptors the
stimulate G-proteins of the Gs class. This increases adenylate cyclase activity and cAMP levels. When inhibitory agonixts (Ai)bind with their
receptors the stimulate G-proteins of the Gi class. This decreases adenylate cyclase activity and cAMP levels. cAMP exerts its effect by
activating protein kinase A (PKA) which phosphorylate proteins, e.g. enzymes and pumps, and in turn increases or decreases their activity.


          A.         cAMP is made by a family to membrane spanning enzymes called collectively Adenylate
                     Cyclase.

                     1.         Currently types I through IV have been well characterized. Additional types
                                probably exist.

                     2.         An ATP-Mg++ complex is the substrate and free Mg++ is a necessary cofactor.

          B.         Receptors which associate with G-proteins of the Gs-type stimulate adenylate cyclase.
                     Receptors which associate with G-proteins of the Gi-type inhibit adenylate cyclase.

          C.         The cAMP that is formed activates “cAMP-dependent protein kinase” (also called
                     Protein Kinase A or PKA).

          D.         PKA phosphorylates other proteins (enzymes, transporters, etc.) and, depending on the
                     protein, increases or decreases that protein’s activity.

          E.         The activity of adenylate cyclase activity can be modulated by factors other than G-protein
                     binding. Such modulation allows integration of multiple second messenger systems.

                     1.         Kinases (including both PKA and PKC) can phosphorylate adenylate cyclase in
                                some cells. Phosphorylation alters adenylate cyclase activity in those cells.

                     2.         Binding of adenylate cyclase by the  -subunits of G-proteins or Ca++/calmodulin
                                complexes can alter adenylate cyclase activity in a manner that depends on
                                adenylate cyclase type.
                                                                 Membrane Function: Signal Transduction Page 5


Table 2
                                 Type               -Effect         Ca++/Calmodulin
                                                                           Ef f ect
                                   I                 -                      +
                                   II                +                      0
                                  III                0                      +
                                  IV                 +                      0


          F.   Phosphodiesterases break down cAMP and terminate its activity.                  Phosphodiesterase
               activity is also subject to control.

III.      THE PHOSPHOINOSITOL SECOND MESSENGER SYSTEM: IP3 AND DAG

                                                                                        Ca++
                                  A




                                 R           PLC
                                        Gq
                                                                       DAG

                                               PIP2                         PKC

                                                                        Protein
                                                                 IP3              Protein-P


                                                                Endoplasmic Reticulum
                                                     Ca++
                                                          Figure 4
          A.   Phospholipase C (PLC) is a membrane bound enzyme that converts phosphatidyl inositol
               (1.4)bis-phosphate (PIP2) into inositol (1,4,5)tris-phosphate (IP3) and diacylglycerol
               (DAG).

          B.   Phospholipase C is activated when a receptor-ligand complex activates a G-protein of the
               Gq family.

          C.   The IP3 formed at the plasma membrane binds to IP3 receptors on the endoplasmic
               reticulum membrane and releases intracellular Ca++ stores.

          D.   DAG remains membrane associated. Protein kinase C (PKC) translocates from the cytosol
               to the membrane and becomes activated by DAG. The activated PKC, in turn,
               phosphorylates other proteins and alters their function state.

          E.   Activation of the phospholipase C system also causes the influx of Ca++.

          F.   Calcium for both extracellular and sequestered intracellular sources binds one of a family
               of Ca++ binding proteins. This complex binds to yet other proteins and changes their
               functional activity.
Page 6 Membrane Function: Signal Transduction


IV.       GUANYLATE CYCLASE: cGMP AND NITRIC OXIDE AS SECOND MESSENGERS




                   Intracellular                                                                  Membrane Bound
                   Ca++ Stores              Ca++                    C.M.
                                                                                                  Guanylate Cyclase
                                                       Ca++
                                                                      NO
                                                                                     GTP

                         NO             Ca++            NO
                                                                                                       Soluble
                                                                                                   Guanylate Cyclase
                      Synthetase
                                                      +
                                                  Citrulline GTP                     PDE
                                                                      cGMP                    GMP
                          Arginine
                                                                  Ion Channels
                                                               cGMP-Dependent PK
                                                                 PDEase Activity

Figure 5. Three second messangers: Ca++, Nitric oxide (NO), and cGMP. Increased intracelluar Ca++ can occur through receptor operated
channels or by release of intracellular calcium stores. Calcium binds with a calcium binding protein such as calmodulin (C.M.), and this
complex in turn activates Nitric Oxide Synthatase (NOS). NOS produces nitric oxide (NO) from the amino acid arginine. The NO that is prduced
acivates a soluble form of guanylate cyclase to make cGMP. cGMP levels can also be increaed by receptor activation of a membrane bound for
of the guanylate cyclase enzyme. cGMP has a variety of tissue-specific effects.


          A.         Membrane bound guanylate cyclase appears to be directly coupled to receptors and forms
                     cGMP from GTP when receptors become occupied.

          B.         NO if formed when the amino acid arginine is broken down into NO and citrulline by
                     nitric oxide synthetase. NO synthetase is activated by Ca/calmodulin complex.

          C.         NO exerts its effect by activating a soluble, cytosolic type of guanylate cyclase and thus
                     increasing cGMP.

          D.         NO is an unique second messenger because it is membrane soluble. This allows it to
                     diffuse to nearby cells and increase cGMP levels in those cells as well. Such a phenomena
                     occurs between vascular endothelial cells and nearby smooth muscle cells.

          E.         cGMP exerts its effect in a variety of ways: direct binding to ion channels, altering the
                     activity of some phosphodiesterases, and by activating a cGMP-dependent protein kinase.

          F.         Phosphodiesterases break down cGMP and terminate its action.

V.        SIGNALING BY ACETYLCHOLINE

          A.         Acetylcholine is a neurotransmitter in both the central and peripheral nervous systems. It
                     binds to two broad classes of receptors: nicotinic receptors and muscarinic receptors.

          B.         Nicotinic receptors are composed by 5 subunits: 2 ,  , , and . The subunits are
                     arranged to form a central cavity that extends across the membrane.
                                                Membrane Function: Signal Transduction Page 7



                              Pore
                                                             Agonist
                                                             Binding
                                                               Site




                                                            Gate

                         Figure 6: Nicotinic Acetylcholine Receptor


C.   The core of the nicotinic receptor protein is normally too small for ion passage. When
     ACh binds to the receptor recognition sites on the -subunits a conformational change
     allows cations to cross the membrane through the central receptor core.

D.   Nicotinic receptors on skeletal muscle (designated N1, or Nm) have different  and 
     subunits than nicotinic receptors on autonomic ganglia (designated N2 or Ng). N1 and N2
     are each receptor gene product families not unique receptor types.

E.   Ligand gated channels for other neurotransmitters like GABA, glycine, 5-HT, and
     glutamate have structural and sequence similarity to nicotinic receptors.

F.   Muscarinic receptors, in contrast, are not channels despite being activated by ACh.
     Muscarinic receptors operate through G-proteins to alter the activity of a number of
     second messenger systems.

G.   To date 5 muscarinic subtypes have been cloned and sequenced (m1, m2, m3, m4, m5).

     1.     m1, m3, and m5 receptors appear to activate phospholipase C through Gq. PLC
            activation leads to a rise in intracellular Ca++ (through IP3 & possibly activation of
            plasma membrane Ca++ channels. The rise in intracellular Ca++ can secondarily
            activate Ca++-sensitive K+ and Cl- channels.

     2.     m2 and m4 receptors couple through Gi to inhibit adenylate cyclase. These
            receptors also probably mediate direct G-protein linked (Go or Gi) regulation of
            certain Ca++ and K+ channels.
Page 8 Membrane Function: Signal Transduction


VI.    SIGNALING BY EPINEPHRINE AND                        NOREPINEPHRINE             AND     COUPLING
       THROUGH ADRENERGIC RECEPTORS

       A.     Adrenergic substances bind to three families of receptors:  -receptors, -receptors, and
              2-receptors. Note that 1-receptors and 2-receptors represent different families;
              whereas, all  -receptor subtypes ( 1,  2,  3)belong to a single family. There are currently
              three known subtypes of 1-receptors and three known subtypes of 2-receptors.

       B.     All adrenergic receptors are coupled through G-proteins.

       C.     Occupation of  -receptors stimulates adenylate cyclase to produce cAMP and activate
              PKA. This is Gs-mediated.

       D.     Occupation of 1-receptors increases IP3 and DAG for some subtypes (1B), and activates
              Ca++-channels for other subtypes (1A). Presumably these effects are Gq-mediated and
              involve PLC activation.

       E.     Occupation of 2-receptors leads to inhibition of adenylate cyclase. Presumably these
              effects are Gi-mediated.
                                                               Membrane Function: Signal Transduction Page 9


                    Study Questions for Membrane Function: Signal Transduction
1. What class of signaling molecules activate membrane associated tyrosine kinase? What class activates
   cytosolic receptors?

2. Which subunit of a G-protein confers activity that is fairly specific to a given second messenger
   system? Which G-protein families interact with adenylate cyclase? Which G-protein family interacts
   with phospholipase-C (PLC)?

3. In order for a G-protein dependent response to occur, what molecule must bind to the G-protein in
   addition to the signal molecule/receptor complex?

4. What cellular protein does cAMP directly affect? By what mechanism does this ultimately change
   cellular function?

5. What molecule(s) are produced by phospholipase C activation? By what mechanisms are intracellular
   calcium levels consequently changed? By what mechanisms is intracellular protein activity changed?

6. What “second messenger” molecule is synthesized in response to enzymatic activation by a
   Ca++/calmodulin complex and produces a different second messenger in adjacent cells?

7. Name three ways cGMP acts as a second messenger to alter cell function.

8. What are the two major classes of cholinergic receptor? What are the three major “families” of
   adrenergic receptors?

9. Fill in signal transduction processes for the receptor types listed in the table below.

                                   Receptor                     Transduction Proces
                                  Nicotinic    N1 or Nm
                                               N2 or Ng
                                  Muscarinic   m1, m3, m5
                                               m2, m4
                                  Beta          1,  2,  3
                                  Alpha1       1A, 1B, 1C    Unknown
                                  Alpha2       2A, 2B, 2C

								
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