Molecular Cell Biology Lecture 21 Notes

Molecular Cell Biology Lecture Notes

Lecture #21: G Protein-Coupled Receptors II
Instructor: Mitra Esfandiarei, Ph.D.
Email: mesfan@midwestern.edu
Date: October 13, 2025

Learning Objectives

• Be able to explain how adrenaline can induce the activation of Gqα in smooth muscle cells.

• If given an appropriate figure, be able to trace the activation of GPCR in response to adrenaline in smooth muscle cells, identifying the ligand, receptor, effector proteins, second messenger(s), and their downstream effects.

• Be able to describe the relationship between PLC, IP3, DAG.

• Understand how IP3 production leads to increased cytoplasmic calcium.

• Be able to explain the interaction between Ca²⁺, calmodulin, and CaM-kinase II.

• Understand how active CaM-kinase II controls smooth muscle contraction.

• Be able to explain the role of MLCK and MLCP during smooth muscle contraction.

• Understand the role that PKC plays in regulating smooth muscle contraction.

• Be able to explain the impact of PKC, caldesmon, and MLCP in smooth muscle cells.

• Understand the mechanism for phototransduction and the involvement of the GPCR and the effector protein PDE in the process.

• Understand the differences between Rod and Cone cells in the human retina.

• Understand the role that glutamate release from Rod cells plays in phototransduction.

• Be able to explain the role of Off-Center and On-Center polar cells in night vision.

The Effector Proteins of GPCRs

1. Adenylyl Cyclase

   • Catalyzes the conversion of ATP to 3',5'-cyclin AMP (cAMP) and pyrophosphate.

2. Phospholipase C (PLC)

   • Hydrolyzes inositol phospholipids to inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG).

3. Cyclic GMP-Phosphodiesterase (PDE)

   • Breaks the phosphodiester bond in cGMP to create 5' GMP.

Signaling Through Phospholipase C (PLC)

Smooth Muscle Contraction

• Example: Smooth Muscle Contraction

• Ligand: Adrenaline (Epinephrine)

• Target Cells: Smooth Muscle (SM)

• Receptors: α1-adrenergic receptor (GPCR)

• G Protein: Gqα

• Effector Protein: Phospholipase C (PLC)

• Outcomes: Vasoconstriction, Blood Pressure Increase

Mechanism of Gqα Activation

• The activation of Gqα-coupled α1-adrenergic receptors is crucial for vasoconstriction, which regulates blood pressure during stress or exercise.

• Clinical Application: α1-adrenergic antagonists (e.g., prazosin, terazosin) are antihypertensive agents that relax blood vessels and reduce blood pressure by blocking the Gαq-mediated pathway.

Adrenaline Stimulation of SMC Contraction: Step I

• In smooth muscle cells, adrenaline activates GPCR, leading to the activation of G protein subunit Gqα, which activates effector protein PLC.

• Activated PLC hydrolyzes membrane inositol phospholipids to produce inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG).

• Both DAG & IP3 are identified as second messengers.

Adrenaline Stimulation of SMC Contraction: Step II

• IP3 Mechanism:

  • IP3 binds to IP3 receptor (IP3R) on the sarco/endoplasmic reticulum membrane.

  • This binding opens IP3R, which acts as a ligand-gated Ca²⁺ channel.

  • Ca²⁺ is released into the cytoplasm of smooth muscle cells, increasing cytoplasmic Ca²⁺ and leading to smooth muscle contraction.

• DAG & PKC:

  • DAG remains on the plasma membrane and, together with Ca²⁺, activates the cytoplasmic enzyme protein kinase C (PKC).

Calcium Regulation in SMC Contraction

Calmodulin Activation

• In the cytoplasm, calmodulin has a very high affinity for Ca²⁺.

• At a low concentration of cytoplasmic Ca²⁺, calmodulin is inactive.

• When cytoplasmic Ca²⁺ concentration increases, calmodulin binds to Ca²⁺ and becomes activated.

• The active calmodulin-Ca²⁺ complex targets the cytoplasmic kinase calmodulin (CaM) kinase-II.

Activation and Role of CaM-kinase II

1. The Ca²⁺/calmodulin complex binds to the inhibitory domain of CaM-kinase II.

2. Conformational changes in CaM-kinase II lead to the partial activation of its catalytic domain.

3. The activated catalytic domain undergoes auto-phosphorylation, fully activating CaM-kinase II.

4. Notably, when Ca²⁺ concentration drops, CaM-kinase II remains partially active (calcium-independent phase) until it is de-phosphorylated and fully inactivated by a protein phosphatase.

CaM-kinase II's Role in Muscle Contraction

• Active CaM-kinase II activates MLCK, which phosphorylates the regulatory light chain of myosin.

• This phosphorylation enables myosin to interact with actin filaments, initiating smooth muscle contraction.

• Myosin Light Chain Phosphatase (MLCP):

  • MLCP dephosphorylates myosin light chains, reversing MLCK’s action.

  • This dephosphorylation reduces the affinity of myosin heads for actin, inhibiting cross-bridge formation and allowing relaxation of smooth muscle.

• Balance:

  • The interplay between MLCK activity and MLCP activity determines the extent of muscle contraction or relaxation in smooth muscle cells.

Role of Protein Kinase C (PKC) in SMC Contraction

1. Activation of MLCK:

   • Activated PKC influences MLC phosphorylation.

2. Crosstalk with MAPK Pathway:

   • PKC interacts with the MAPK pathway to enhance contraction.

3. Caldesmon Interaction:

   • PKC promotes the dissociation of caldesmon from actin filaments, exposing myosin-binding sites and allowing increased contraction.

Cyclic Nucleotide Phosphodiesterases (PDEs)

• Definition: PDEs are a group of enzymes that degrade the phosphodiester bond in cAMP and cGMP, regulating the duration, amplitude, and localization of cyclic nucleotide signaling.

• Classification: There are 12 families of PDEs (PDE1-PDE12), categorized based on amino acid sequences, substrate specificities, regulatory proteins, and tissue distributions.

Visual Photo-Transduction

Overview

• Process: Light photons stimulate rod photoreceptor cells through a GPCR (Rhodopsin).

• G Protein: Gtα (transducin) functions in this visual process.

• Effector Protein: Phosphodiesterase (PDE-6)

• Outcome: Vision in dim light.

• Distinction: Rods are sensitive to low light, while cones function in bright light and color vision.

Mechanism of Light Perception

1. Light photons activate rhodopsin receptors in rod cells.

2. Active rhodopsin acts as a guanine nucleotide exchange factor (GEF), activating Gtα.

3. Gtα activates PDE-6, which hydrolyzes cGMP to 5'-GMP, causing a decrease in cytoplasmic cGMP.

4. Lowered cGMP levels lead to the closure of cGMP-gated cation channels, resulting in rod cell hyperpolarization.

5. Hyperpolarization shuts voltage-gated Ca²⁺ channels, decreasing cytosolic Ca²⁺ concentrations.

6. Ca²⁺ is necessary for the release of glutamate from rod cells; reduced Ca²⁺ release results in decreased glutamate levels in the synaptic junction with bipolar cells.

7. Depending on bipolar cell type (ON or OFF), the change in glutamate release activates or inhibits these cells, facilitating night vision.

On-Center and Off-Center Bipolar Cells

• Dark Adaptation:

  • In darkness, high cGMP levels keep cation channels open; rod cells release glutamate, leading to depolarization of OFF-center bipolar cells and hyperpolarization of ON-center bipolar cells (no firing).

• Light Adaptation:

  • In light, low cGMP closes cation channels; rods do not release glutamate. This inactivity causes ON-center bipolar cells to depolarize (firing means vision) while OFF-center bipolar cells remain hyperpolarized.

Summary Slide for GPCRs