hormones lecture h03

HormonesG Protein’s SignalingCalcium Signaling

Dr. Aga Syed SameerCSIR Lecturer (Demonstrator)Department of Biochemistry,Medical College,Sher-I-Kashmir Institute of Medical Sciences, Bemina, Srinagar, Kashmir, 190010. India.

First MBBS

Lecture No: H 03

Time : 10:00am

Dated: 10/03/2015

• cGMP is produced by activation of Gualylyl Cyclase via various signal molecules : Nitric Oxide, Guanylin, Atrial Natri-UreticFactors.

• Gualylyl Cyclase is made of three components:• N Terminal Ligand Binding Domain

• TMD of alpha helices

• C terminal Domain of enzymatic activity

cGMP

• Produced by activation of Gualylyl Cyclase via various signal molecules : Nitric Oxide, Guanylin, Atrial Natri-Uretic Factors.

• In Kidneys: cGMP molecules are produce in response to ANF’s.

ANF’s are produced by heart muscles in response to increased blood volume.

ANF’s activates guanylyl cyclase in cells of collecting ducts (Kidney’s)

cGMP triggers increased renal excretion of Na2+ and consequently water (change in osmotic pressure).

cGMP

• In Intestinal Cells:

Guanylin – intestinal peptide activates a similar receptor GC in PM to produce cGMP molecules.

Guanylin – is the regulator of Cl- secretion in the Intestines.

This intestinal GC receptor is important as it is an non conventional target of heat stable endo toxin of E. coli.

Endotoxin produced by E. coli causes a drastic elevation of cGMP(above normal) levels in the intestinal cells.

Net effect is the increased Cl- secretion and consequently decreased reabsorption of water by Intestinal epithelia, leading to diarrheoa.

cGMP

• In Endothelial Cells: cGMP molecules are produced in response to Nitric Oxide (NO).

NO is produced by nitric oxide synthase located on the outer surface of endothelial cells usually in response to acetyl choline.

Acetyl choline binding to endothelial cell surface cause surge of Ca2+

concentration which triggers the NO synthase activity.

NO in turn diffuses across plasma membrane and stimulates guanylylcyclase.

Protein Kinase G is its effector protein via which cGMP mediates its function by phosphorylating Ser and Thr residues in target proteins.

cGMP

Protein Kinase G is its effector protein via which cGMPmediates its function.

It is a single polypeptide possessing both catalytic and regulatory domains of the enzyme.

On binding with cGMP forces the regulatory domain out of catalytic domain.

It causes phosphorylation Ser and Thr residues in target proteins.

Protein Kinase G - PKG

• Phospholipids of the membranes are converted into second messengers by a variety of Enzymes that are regulated in response to extracellular signals.

• These enzymes include:

Phospholipases (Lipid Splitting Enzymes)

Phospholipid Kinases (Lipid Phosphorylating Enzymes)

Phospholipid Phosphatases (Lipid De-phosphorylating Enzymes)

• The “Phospholipases” hydrolyse specific ester bonds that connect the different building blocks of the phospholipid molecule.

Phosphatidyl Inositol Derived Second Messengers

Phosphatidyl Inositol Derived Second Messengers

• It is specific to Phosphatidyl Inositol.

• PLC β 1 is inturn activated by Gq of the trimeric G proteins.

• It is situated in the inner surface of the membrane; bound by interactions between its PH domain and a phosphoinositideembedded in the bilayer.

• PLC β on activation; catalyses a reaction that splits PIP2 {PI (4,5)P2} into two molecules:

IP3 : Inositol 1,4,5 Triphosphate

DAG : Di Acyl glycerol

Phospholipase C β 1

• It is a sugar phosphate.

• Small water soluble molecule capable of rapid diffusion throughout the interior of cell.

• IP3 molecules formed at the membrane diffuse into the cytoplasm and bind to specific IP3 receptors located at the surface of SER.

• SER is a site of calcium storage in a variety of cells.

• IP3 itself also functions as tetrameric channel for Calcium ions.

Inositol 1,4,5 Triphosphate

• SER channels are henceforth opened up allowing calcium ions to diffuse into cytoplasm.

• Calcium ions then act as second messengers by binding to various target molecules – triggering specific responses:

Contraction of smooth muscle cells

Exocytosis of histamine- containing secretory granules


• It is a Lipid molecule.

• Attaches itself to DAG Binding C1 domain of effector protein.

• Mediates its function via Protein Kinase C usually.

• Protein Kinase C phosphorylates Ser/Thr residues in wide variety of proteins.

• Protein Kinase C plays critical role in cellular growth and differentiation; Cellular metabolism; Cell death; transcriptional activity.

Di Acyl Glycerol

Calcium Signaling

Calcium

• Extracellular calcium concentration is 5mM/L

• Intracellular free/ionized calcium concentration is very low ~0.05-10.0 uM/L

• Substantial amounts of calcium are associated with intracellular organelles such as Endoplasmic Reticulum & Mitochondria

• Inspite of this large concentration gradient & a favorable trans-membrane electrical gradient ; calcium is restricted from entering the cell

Calcium Homeostasis• Four calcium channels (mainly) play a critical role in

regulating the calcium levels in the cytosol

• Two are highly functional in basal conditions

• Na+/Ca2+ channel in plasma membrane

• SERCA in Smooth Endoplasmic Reticulum

• Two are activated and in use under hormone or neurotransmitter action

• Voltage dependent Ca2+ channels in Plasma Membrane

• Ryanodine Receptors in Smooth Endoplasmic Reticulum

• There are three ways of changing the cytosolic calcium concentration:

By activating the receptors that serves as the channels (ligandgated Ca2+ channels) in the plasma membrane of the cell by hormone signaling.

Indirectly promoting the calcium influx by modulating the membrane potential at the plasma membrane, inturn opens the voltage gated Ca2+ channels.

Ca2+ can be mobilized from ER and also from mitochondrial parts

• Hormone action via Ca2+ involves the mediation of intracellular targets of Ca2+ ; which are Ca2+ - dependent regulator of “phosphodi-esterase /kinase activity”

Calcium Homeostasis

Calcium Homeostasis

Calmodulin – The Mediator• Is a Ca2+ - dependent regulatory protein

• 17kDA protein: Homologous to muscle protein troponin C is structure & function

• It has four Ca2+ - binding sites & full occupancy of these sites leads to a marked conformational change

• This allows calmodulin to activate Enzymes & Ion channels

• The interaction of Ca2+ with calmodulin is similar to the binding of cAMP to PKA and the subsequent activation of this molecule

• Calmodulin then mediates its action via CaM Kinase

Calmodulin

• Calmodulin is particularly involved in regulating various Kinases and Enzymes of cyclic nucleotide generation & degradation

• In addition, Ca2+/Calmodulin regulates the activity of many structural elements in cells:

• Actin-Myosin complex of smooth muscles

• Micro-filament mediated processes

• Mitosis

• Granule release

• Endocytosis

Calmodulin

• Number of critical metabolic enzymes are regulated by Ca2+, Phosphorylation /Both:

Glycogen Synthase;

Pyruvate Kinase;

Pyruvate Carboxylase;

Pyruvate Dehydrogenase;

Glycerol 3 Phosphate Dehydrogenase

Signaling• Hormone/Ligand activates GPCRs and Gq which inturn

activates PLCβ1

• IP3 and DAG activate Protein Kinase C

• PKC activity is inturn affected by calcium concentration in cytosol

• IP3 interacts with specific receptors (Ryanodine Receptors) and release calcium from SER into cytoplasm

• The activation of PKC and Increased calcium concentration activates “Ca2+ - Calmodulin Dependent Kinases (CaMK)” which mediates its effect Via CREB (cAMP Response element Binding- Protein)

Ca2+/Calmodulin

Questions?