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Research in Signal Transduction

Caffeine

Caffeine clearly produces a state of arousal in the central nervous system. High levels appear to block the binding of an inhibitory neurotransmitter, adenosine, to the A2A adenosine receptor. In the absence of caffeine, adenosine levels rise during the day, which promotes interaction with its receptor, leading to increasing sleepiness and lack of concentration. When adenosine binds normally to its receptors, it activates the adenylate cyclase cascade, which activates PKA, leading to changes in phosphorylation state of many proteins inside the cell, including protein phosphatase (2A). These changes inhibit neural firing. Caffeine blocks these changes. 

Allosterism and Signal Transduction

In a recent review article, Changeux and Edelstein review the MHC model 40 years after its conception and support its application to signal transduction processes. They include in siganling molecules not only hemoglobin, but regulatory enyzmes (aspartate transcarbamylase, phosphofructokinase, LDH, glycogen phosphorylase), membrane receptors (acetylcholine receptor, rhodopsin), and nuclear receptors (lac repressor, steroid hormone receptors). In all these signaling proteins, residue distant from the "active" site participate in binding to allosteric ligands. Often the allosteric site is on a separate domain which can be cleaved from the protein and still maintain allosteric ligand binding properties. The proteins also consists of multiple subunits easily related by distinct symmetry axes. Allosteric ligands often bind in cavity insubunit interfaces along symmetry axes. In general, crystal structure analyses show that low affinity T and high affinity R forms of the signaling proteins exists, but accompanied by minor tertiary structure changes in individual subunits (i.e. perfect symmetry in all subunits is not preserved on binding of allosteric ligand). For neurotransmitter membrane receptors, these two states can be correlated with an open and closed state (for ion flux), and open conformations of these proteins can often be found in mutant forms. However, for many ligand-gated ion channels and G-protein coupled receptors (serpentine), kinetic analyses show more complicated forms than can be represented by a simple two state (R and T) model. High-resolution microscopy shows evidence for nonsymmetrical quaternary structural changes. These change can be observed in the absence of ligand, which gives support to the MWC concept that allosteric ligands select certain conformational states, leading to equilibrium shifts in the unliganded receptor to the more high affinity state. More refined methods of structural analysis will presumably show more evidence of subtle tertiary changes in the proteins that are preludes to quaternary structural changes. Yet the simplicity of the MWC model for explaining many features of signaling proteins remains.

The Human Genome and Signal Transduction

With the determination and annotation of the human genome, it has become very clear that a significant fraction of the human genome (about 40% of the 58% of known genes determined by Venter et. al. and published in Science, 291, 1335, 2001) is devoted directly or indirectly to signal transduction processes. These include signal molecules, receptors, kinases, regulators, protooncogenes and ion channels. The chart below shows the relative distribution of over 26,000 genes of known function (with 42% still of unknown function.

Figure:Distribution of Molecular Functions of 26,383 Genes

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Signal Transduction and Lipid Rafts

When extracellular signals bind to membrane receptors, conformational changes in the receptor protein signals the inside of the cells that the receptor is bound with a ligand. Once bound, the receptor often move in the membrane and clusters in outer leaflet rafts that contain cholesterol and spingholipids, with longer and more saturated fatty acids. This increases the thickness and decreases the fluidity of the bilayer in the raft. Inner leaftlet rafts are also observed. Caveolae (regions of the membrane that are invaginated) and to which the protein caveolin is bound, are found in both leaftlets. Using fluorescent microscopy, Zacharias et. al. were able to detect movement of specific proteins into raft regions. Proteins that are covalently modified with a fatty acid (myristic and palmitic acid) move to the caveolae while isoprene-modified proteins (such as the the prenyl group geranylgeranyl) do not move to the caveolae.  

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New Research

Schizophrenia is a complex brain disease that has been linked to alterations in neuronal brain signaling by many different neurotransmitters, including serotonin (5HT), dopamine, and glutamate. Illicit drugs that also interact with the receptors for those neurotransmitter (LSD, amphetamne, and phenylcyclidine or PCP, respectively) also can induce symptoms characteristic of schizophrenia. Might these three different drugs, and their cognate neurotransmitters, induce signal transduction mediated by a common protein? Sveningsoon, Greengard, et al. have found such a common intermediary protein called DARPP-32, which regulates the activity of signal transduction kinases/phosphatases in these different neurons. 

To test their hypothesis that these drug signal produce effects which overlap at DARPP-32, they produced knock out mice lacking this gene and investigated the effects of the drugs on these mice. Their "assay" was to look at alteration in behaviors common to schizophrenic and some people on those drugs. In normal mice and people, if several "pulses" of tactile stimulation are given before inducing a startle reflex, the reflex is inhibited. "Prepulse inhibition of a startle reflex is inhibited in schizophrenics and in humans and mice under the influence of these drugs. Also, schizophrenics and drug-treated mice/humans often repeat motions (perseverative behavior). Knock out mice did not experience these behavior effects when given these drugs. 

The DARPP-32 protein has four Ser/Thr residues which can be phosphorylated by kinases (at Thr 34, Thr 75, Ser 97, and Ser 130) and dephosphorylated by phosphatases.The phosphorylations each appear to have different effects:

  • Thr 34 - inhibit protein phoshatase I (PP1)
  • Thr 75 - inhibit protein kinase A (PKA)
  • Ser 97 - activate protein kinase A (PKA) to phosphorylate Thr 34
  • Ser 130 - prevent dephosphorylation of Thr 34 by protein phosphatase 2B (PP2B)

Sveningsson, Greengard, et al. investigated the effects of LSD, amphetamine, and PCP on the phosphorylation state of these amino acid side chains in DARPP-32. Their study showed that the ultimate effect of these drugs affect the phosphorylation state of Thr 34, Thr 75, and Ser 130 in fashion that inhibits PP1 activity. (All three drugs increased phosphorylation of Thr 34 and Ser 130, and amphetamine and LSD decreased Thr 75 phosphorylation.) The activity of proteins downstream in the signaling cascade from PP1 (specifically glycogen synthase kinase 3 - GSK-30 , cAMP response element binding protein - CREB) and c-fos) are then effected. 

Figure:Effect LSD, PCP, and amphetamines on signaling

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Long Term Memory - Long Term Potentiation (LTP)

The conversion of short term to long term memory must produce long-lasting changes in molecular structure in neurons associated with the memory. The affected neurons presumably would be induced to increase their response to a stimuli. In addition, a mechanism must exist to maintain the "potentiated" state of the altered neuron. These combined processes are termed long term potentiation (LTP). Until recently, little was known about the maintenance phase of LTP. Pastalkova et al. have shown than in hippocampal (a part of the brain required for conversion of short term to long term memory), maintenance of LTP and long term memory involving spatial learning requires the persistent activation of a particular isozyme of protein kinase C called Mzeta (PKMz). These investigators made a peptide inhibitor of the kinase by synthesizing a small fragment of the actual kinase (which must have had enough structure to compete with binding of PKMz to its target substrates). The inhibitor, named ZIP, was injected into rat hippocampus, and compared to controls (saline or a scrambled version of the inhibitor) reversed LTP maintenance and caused loss of 1-day old spatial memory.