GPCR Signaling and Nobel Laureates

Proteins Associated with GPCRs

• Beta-arrestins
• AP-2
• Clathrin
• Dynamin

Desensitization, Endocytosis, and Recycling of GPCRs

• These processes regulate GPCR signaling.

Learning Objectives

• Discuss the role of proteins associated with GPCRs, focusing on beta-arrestins and clathrin.
• Introduce types of receptor desensitization and key players involved.
• Emphasize the functional implications of clathrin-mediated endocytosis on GPCR signaling.

Historical Background of Arrestins

• 1986: Wilden et al. isolated visual arrestin.
• 1987: Benovic et al. increased purification of GRK2 (beta-ARK1) from bovine brain, revealing a missing cofactor for receptor desensitization. Adding visual arrestin restored the desensitizing ability and the missing protein was called beta-arrestin 1.
• 1992: Attramadal et al. cloned beta-arrestin 2.

General Features of Arrestins

• Arrestin is involved in G protein-coupled receptor signaling.
• Binds to the C-terminal (or 3rd intracellular loop (IC3)) of GPCRs following phosphorylation by GRK at Ser/Thr residues.
• Arrestins lack intrinsic enzymatic activity.

Four Functional Members of the Arrestin Family

• Two arrestins: Visual arrestin (45 kDa, first identified) and Cone arrestin, almost exclusive to the retina, regulating photoreceptor function.
• Two beta-arrestins: Beta-arrestin 1 and 2, are ubiquitous proteins, primarily found in the brain and spleen.

Structural Domains of Arrestins

• Includes N-terminal regulatory domain, proline-rich region, and C-terminal regulatory domain; also contains secondary receptor binding domain; phosphate sensor domain; and binding domains for clathrin and AP2.

Functions of Arrestin

• Role in GPCR desensitization and endocytosis.
• Acts as a scaffold protein to:
  • Facilitate signal pathway activation
  • Interact with MAP kinases, PI3-kinases, PKB/Akt, and Src.
  • Lead to ERK activation and endocytosis.
  • Interact with MAPK kinase kinases (e.g., ASK1) and JNK3, leading to activation of the MAPK JNK3 signaling.

Multiple Actions of Beta-Arrestin

• Shows interaction with effector molecules, GRK2, and phosphorylation, leading to endocytosis.
• Involved in ERK activation and endocytosis, JNK activation, and related processes.

AP-2

• A clathrin-adaptor protein complex of 4 adaptins (α-, β2-, μ2-, and σ2-adaptins).
• Associates with the plasma membrane.
• Aids in endocytosis.

Clathrin

• A protein forming a three-legged structure called a triskelion.
• Composed of 3 light chains and 3 heavy chains, each with two domains (proximal and distal) and an N-terminal domain acting as a binding site for assembly particles and a trimerization domain.
• Assembles into a polyhedral lattice (soccer-ball shaped) serving as a scaffold for vesicle assembly and stabilizing mature vesicles during transport.
• Interacts with AP-2 through its terminal domain.

Dynamin

• A cytoplasmic GTPase primarily involved in scission of newly formed clathrin-coated vesicles from the membrane.

The 2013 Nobel Prize in Physiology or Medicine

• Awarded to James E. Rothman, Randy W. Schekman, and Thomas C. Südhof for their discoveries of machinery regulating vesicle traffic.

Regulation of GPCR Signaling

• Includes endocytosis, desensitization/resensitization, downregulation/upregulation, and recycling of GPCRs.

Patterns of Desensitization

• Homologous Desensitization: Agonist attenuating response to that agonist through its receptor.
• Heterologous Desensitization: Agonist attenuating responses to other receptors in the cell. Induced by activation of one type of GPCR (heterologous).

Desensitization

• Homologous and Heterologous types; distinguished by whether activation of one receptor influences the response of another.
• Receptor phosphorylation by GPCR kinases (GRKs).
• Beta-arrestin-mediated (usually followed by clathrin-mediated endocytosis).
• Beta-arrestin-independent (receptor further phosphorylated by GRKs to undergo subsequent clathrin-mediated endocytosis.)

Homologous Desensitization

• Characterized by Ligand binding, receptor phosphorylation, beta-arrestin recruitment and binding, clathrin and AP-2 recruitment leading to endocytosis.
• Associated downstream signaling is altered because of internalization

Beta-arrestin-mediated B2AR Homologous Desensitization

• Only occurs when the agonist is bound to the receptor (active)
• GRK phosphorylation of GPCR increases affinity of the receptor to beta-arrestin to impair coupling to G-proteins.
• Binding of beta-arrestin to the phosphorylated receptor occurs only in GRK-dependent phosphorylation.

PKA-Induced Heterologous GPCR Desensitization

• PKA phosphorylates B2-adrenergic receptors.
• Diverts coupling from G to Gαs.
• Activation of βγ subunits leads to ERK and nuclear responses.
• Desensitization of newly coupled Gαs signaling occurs via GRK-mediated phosphorylation.

Heterologous Desensitization of β2-AR

• Involves both PKA- and GRK-mediated phosphorylations.

PKA-mediated β2AR Desensitization

• A negative feedback mechanism.
• PKA can phosphorylate inactive B2ARs.
• PKA activity stimulated by receptors promoting adenylyl cyclase activation.

Regulation of GPCR Signaling through Desensitization/Resensitization

• Desensitization: Rapid reduction in receptor signaling capacity within minutes.
• Resensitization: Rapid reactivation of receptors in response to agonist action.
• Desensitization/Resensitization, downregulation and upregulation are important for physiological regulation.

Involvement of GPCR Recycling in Receptor Resensitization

• This involves ligand binding, receptor phosphorylation, beta-arrestin recruitment, clathrin and AP2 recruitment, endocytosis, lysosomal degradation, receptor ubiquitination, and recycling to the endosomal compartment.

Regulation of GPCR Signaling through Downregulation/Upregulation

• Downregulation: Reduction in the total number of receptor sites over hours after prolonged agonist activation.
• Upregulation: A relatively slow increase in the number of receptors.

Determinants for GPCR Fate: Recycling Versus Degradation

• Factors targeting recycling: dephosphorylation, transient β-arrestin interaction, presence of recycling sequences in the C-terminus of GPCRs (e.g., NHERF, NSF.)
• Factors targeting degradation: stable complex formation with beta-arrestin mediated by phosphorylation-dependent clusters in C-terminus, ubiquitination of beta-arrestins and GPCRs.

Functional Consequences of Clathrin-mediated Endocytosis

• Role in rapid desensitization, resensitization, proteolytic downregulation, controlling specificity of signal transduction.

Role of Endocytosis in Controlling the Specificity of Signal Transduction

• Endocytosis involves forming a protein complex on endosome membranes, including internalized GPCRs, signal-transducing kinases (like c-Src), or receptor tyrosine kinases (like EGFRs), co-endocytosed from the plasma membrane.

Test your knowledge on the discoveries related to vesicle transport and GPCR signaling. This quiz covers the roles of key Nobel Laureates and the mechanisms of regulation in cell signaling. Explore the functions of proteins like β-arrestins and their impact on receptor desensitization.