G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes.
single polypeptide chain comprising of seven transmembrane α-helices
extracellular N-terminal domain of varying length,
intracellular C-terminal domain.
length of the extracellular N terminus and the location of the agonist binding domain determines family.
The long, third cytoplasmic loop couples to the G-protein
Usually particular receptor subtypes couple selectively with particular G-proteins
For small molecules, such as noradrenaline, the ligand-binding domain of class A receptors is buried in the cleft between the α-helical segments within the membrane. Peptide ligands bind more superficially to the extracellular loops
G protein system
GPCRs interact with G proteins in the plasma membrane when an external signaling molecule binds to a GPCR, causes a conformational change in the GPCR.
G-proteins comprise a family of membrane-resident proteins whose function is to recognise activated GPCRs and pass on the message to the effector systems that generate a cellular response.
G proteins are specialized proteins with the ability to bind the nucleotides guanosine triphosphate (GTP) and guanosine diphosphate (GDP).
The G proteins that associate with GPCRs are heterotrimeric, (alpha beta and gamma subunits)
alpha and gamma are attached to the plasma membrane by lipid anchors
Trimer in resting state
activated alpha monomer and beta/gamma dimer
Guanine nucleotides bind to the α subunit, which has enzymic activity,catalysing the conversion of GTP to GDP. The β and γ subunits remain together as a βγ complex. All three subunits are anchored to the membrane through a fatty acid chain, coupled to the G-protein through a reaction known as prenylation.
G-proteins are freely diffusible so a single pool of G-protein in a cell can interact with several different receptors and effectors
When GPCR is activated by an agonist, a conformational change causes it to acquire high affinity for αβγ (G protein)
bound GDP dissociates and is replaced with GTP, which in turn causes dissociation of the G-protein trimer, releasing α-GTP and βγ subunits - the ‘active’ forms of the G-protein
which diffuse in the membrane and can associate with various enzymes and ion channels
Signalling is terminated on hydrolysis of GTP to GDP through the GTPase activity of the α subunit.
resulting α–GDP dissociates from the effector, and reunites with βγ
Attachment of the α subunit to an effector molecule increases its GTPase activity
GTP hydrolysis is termination –> activation of the effector tends to be self-limiting
Second messenger targets for G proteins
Adenylyl cyclase (responsible for cAMP formation)
Phospholipase C (inositol phosphate and diacylglycerol (DAG) formation)
Ion channels, particularly calcium and potassium channels
Rho A/Rho kinase (system controlling the activity of many signalling pathways for cell growth and proliferation, smooth muscle contraction, etc.)
Mitogen-activated protein kinase (MAP kinase) system controlling cell functions eg division.
Please note there are 2 more types of signal receptors: Protein Kinase Receptors: transmembrane-protein enzymes that are kinases, that add a phosphate group to protein. The best understood of these are Receptor Tyrosine Kinases. Intracellular Receptors: receptors positioned in the cytoplasm or nucleus, and are able to pass through the phospholipid membrane.
Cyclic adenosine monophosphate (cAMP) is a second messenger important in many biological processes. cAMP is derived from adenosine triphosphate (ATP) and used for intracellular signal transduction in many different organisms, conveying the cAMP-dependent pathway.
cAMP is a second messenger, used for intracellular signal transduction, such as transferring into cells the effects of hormones like glucagon and adrenaline, which cannot pass through the plasma membrane. It is involved in the activation of protein kinases and regulates the effects of adrenaline and glucagon. cAMP also binds to and regulates the function of ion channels such as the HCN channels and a few other cyclic nucleotide-binding proteins such as Epac1 and RAPGEF2.
Discovery links rare, childhood neurodegenerative diseases to common problem in DNA repair
St. Jude Children’s Research Hospital scientists studying two rare, inherited childhood neurodegenerative disorders have identified a new, possibly common source of DNA damage that may play a role in other neurodegenerative diseases, cancer and aging. The findings appear in the current issue of the scientific journal Nature Neuroscience.
Researchers showed for the first time that an enzyme required for normal DNA functioning causes DNA damage in the developing brain. DNA is the molecule found in nearly every cell that carries the instructions needed to assemble and sustain life.
The enzyme is topoisomerase 1 (Top1). Normally, Top1 works by temporarily attaching to and forming a short-lived molecule called a Top1 cleavage complex (Top1cc). Top1ccs cause reversible breaks in one strand of the double-stranded DNA molecule. That prompts DNA to partially unwind, allowing cells to access the DNA molecule in preparation for cell division or to begin production of the proteins that do the work of cells.
Different factors, including the free radicals that are a byproduct of oxygen metabolism, result in Top1ccs becoming trapped on DNA and accumulating in cells. This study, however, is the first to link the buildup to disease. The results also broaden scientific understanding of the mechanisms that maintain brain health.
Investigators made the connection between DNA damage and accumulation of Top1cc while studying DNA repair problems in the rare neurodegenerative disorders ataxia telangiectasia (A-T) and spinocerebellar ataxia with axonal neuropathy 1(SCAN1). The diseases both involve progressive difficulty with walking and other movement. This study showed that A-T and SCAN1 also share the buildup of Top1ccs as a common mechanism of DNA damage. A-T is associated with a range of other health problems, including an increased risk of leukemia, lymphoma and other cancers.
“We are now working to understand how this newly recognized source of DNA damage might contribute to tumor development or the age-related DNA damage in the brain that is associated with neurodegenerative disorders like Alzheimer’s disease,”said co-corresponding author Peter McKinnon, Ph.D., a member of the St. Jude Department of Genetics. The co-corresponding author is Sachin Katyal, Ph.D., of the University of Manitoba Department of Pharmacology and Therapeutics and formerly of St. Jude.
A-T and SCAN1 are caused by mutations in different enzymes involved in DNA repair. Mutations in the ATM protein lead to A-T. Alterations in the Tdp1 protein cause SCAN1.
Working in nerve cells growing in the laboratory and in the nervous system of specially bred mice, researchers showed for the first time that ATM and Tdp1 work cooperatively to repair breaks in DNA. Scientists also demonstrated how the proteins accomplish the task.
The results revealed a new role for ATM in repairing single-strand DNA breaks. Until this study, ATM was linked to double-strand DNA repair. ATM was also known to work exclusively as a protein kinase. Kinases are enzymes that use chemicals called phosphate groups to regulate other proteins.
Scientists reported that when Top1ccs are trapped ATM functions as a protein kinase and alert cells to the DNA damage. But researchers found ATM also serves a more direct role by marking the trapped Top1ccs for degradation by the protein complex cells use to get rid of damaged or unnecessary proteins. ATM accomplishes that task by promoting the addition of certain proteins called ubiquitin and SUMO to the Top1cc surface.
Tdp1 then completes the DNA-repair process by severing the chemical bonds that tether Top1 to DNA.
Mice lacking either Atm or Tdp1 survived with apparently normal neurological function. But compared to normal mice, the animals missing either protein had elevated levels of Top1cc. Those levels rose sharply during periods of rapid brain development and in response to radiation, oxidation and other factors known to cause breaks in DNA.
When researchers knocked out both Atm and Tdp1, Top1cc accumulation rose substantially as did a form of programmed cell death called apoptosis. Investigators reported that apoptosis was concentrated in the developing brain and few mice survived to birth. McKinnon said the results add to evidence that the brain is particularly sensitive to DNA damage.
Researchers then used the anti-cancer drug topotecan to link elevated levels of Top1cc to the cell death and other problems seen in mice lacking Atm and Tdp1. Topotecan works by trapping Top1ccs in tumor cells, resulting in the DNA damage that triggers apoptosis. Investigators showed that the impact of Top1cc accumulation was strikingly similar whether the cause was topotecan or the loss of Atm and Tdp1.
Once chromosomes are pulled to either side, the cell start reversing the steps of prophase and prometaphase: the nuclear envelop reforms around decompacting chromosomes. Near the end of telophase, a thin bridge between the daughter cells, called the midbody contains the remnants of the mitotic spindle. A ring of actin filaments (i.e., the cleavage furrow) pulls like ‘purse strings’ to pinch the cells into two.
Image: Two HeLa cervical cancer cells captured in telophase, as sister chromatids are separated into the two ends of the dumbbell-shaped cell. Green fluorescence is from Aurora B protein kinase fused to eGFP with white and red marking DNA and tubulin. The image was taken using a DeltaVision deconvolution/restoration microscope.
A virtual supercomputer running on more than 239,000 computers around the world has successfully eavesdropped on a protein key to cancer’s progression in the body.
Researchers using Stanford University’s Folding@home, a distributed computational platform, have been able to describe the activation of a protein called Src kinase, a molecular switch that is believed to turn on the tumor-producing signals in cells that tell them to grow, spread and not self-destruct.
The team says it is the first time the protein has been modeled as it changes from an inactive state to an active one. Their insight could help develop new drugs that specifically target Src kinase.
(The gif above illustrates Folding@home’s simulated protein-folding steps from an uncoiled configuration to a complex, 3-D structure. The protein here is NTL9 and unrelated to Src kinase, the subject of this article. See the interesting video below. Courtesy Vijay Pande/Stanford.)