Lecture 7 - Bioc 325 (2023) PDF

Proteins Associated with GPCRs-part2: β-arrestins AP-2 Clathrin Dynamin Desensitization, Endocytosis and Recycling of GPCRs 1 1. Discuss the role of some proteins associated with GPCRs, mainly β-arrestins and clathrin...

Proteins Associated with GPCRs-part2: β-arrestins AP-2 Clathrin Dynamin Desensitization, Endocytosis and Recycling of GPCRs 1 1. Discuss the role of some proteins associated with GPCRs, mainly β-arrestins and clathrin 2. Introduce the types of receptor desensitization, and the important players involved 3. Emphasize the functional implications of clathrin-mediated endocytosis on GPCRs Signaling 2  β-arrestins AP-2 Clathrin Dynamin 3  Wilden et al., 1986: (group of Kuhn H) 1986 Isolation of visual arrestin Benovic et al., 1987: (group of Lefkowitz RJ) Increased purification of GRK2 (βARK1) from bovine brain  lower ability of uncoupling G-protein from β2AR  purification led to a missing cofactor vital for receptor 1987 desensitization Addition of visual arrestin:  restored the desensitizing ability of the purified βARK The missing protein was called β-arrestin1 Attramadal et al., 1992: (group of Lefkowitz RJ) 1992 Cloning of β-arrestin2 4  Nomenclature: Arrestin: Arresting G protein-coupled receptor signaling  Binds to the C-terminal (or 3rd intracellular loop (IC3): site of binding of Gα) of GPCR, following receptor phosphorylation with GRK at Ser/Thr residues  Arrestins lack any intrinsic enzymatic activity 5  Two Two β- arrestins arrestins Visual arrestin (45 kDa, first β-arrestin1 identified) Cone arrestin β-arrestin2 almost exclusive in retina ubiquitous proteins regulate photoreceptor mainly in brain, spleen function 6 Luttrell LM, Lefkowitz RJ. J Cell Sci. 2002;115:455–465.  receptor activation domain secondary C-terminal (site of binding to receptor-binding regulatory activated receptor) domain domain Pro-rich region phosphate N- terminal clathrin and AP-2 (mammalian β- sensor regulatory binding domains arrestin1& 2) domain domain binds to SH3 of cSrc 7 Pharmacol Rev 53:1–24, 2001  Role in GPCR Desensitization and Endocytosis  As a Scaffold 8 1. Role in GPCR Desensitization and Endocytosis ◦ Arrestins play important roles in homologous desensitization of GPCR. ◦ Homologous desensitization is a mechanism leading to the decrease of the ligand-associated signaling of the receptor due to p~ of GPCR by GRK and subsequent uncoupling of G-protein from the receptor by arrestin. ◦ Arrestin binds to the GRK-phosphorylated receptor and not only uncouples the receptor from its G protein, but also acts as a scaffold to recruit the endocytotic machinery proteins, clathrin and the clathrin adaptor:AP2, which target the activated receptor to clathrin-coated pits receptor endocytosis. 9 2. As a scaffold protein forming a complex with a range of signaling molecules  facilitating signal pathway activation. Examples include 1. MAP kinases (ERK and JNK) 2. PI3-kinase 3. PKB/Akt 4. Src ◦ Arrestin interacts with Src leading to ERK activation and endocytosis ◦ Arrestin interacts with a MAPK kinase kinase (for example, ASK1) and c-Jun N-terminal kinase 3 (JNK3) thus leading to activation of the MAPK JNK3 signaling 10  3 1 2 11 Rockman et al. 2002 NATURE, VOL 415   Name: Clathrin-Adaptor protein complex-2  Stable complex of 4 adaptins: 1. α-adaptin 2. β2-adaptin 3. μ2-adaptin 4. σ2-adaptin It associates to the plasma membrane  aids in endocytosis 12 http://www.endocytosis.org/Adaptors/  Clathrin is a protein with (a) Triskelion Structure of Clathrin a three-legged structure called triskelion Distal domain  It is made of: ◦ 3 light chains ◦ 3 heavy chains:  each having:  2 domains: proximal and distal  An N-terminal domain: Binding site for assembly particles Trimerization Proximal ◦ A trimerization domain: domain linking light chains with domain heavy chains 13  Clathrin Clathrin molecules assemble in a polyhedral (soccer-ball- shaped) lattice that serves two roles: 1. a scaffold for the assembly of vesicles 2. a protein coat that stabilizes the mature vesicle during transport to the site of fusion. 14  A clathrin triskelion is shown interacting through its terminal domain (TD) with the appendage domains of the β2 subunit of adaptor protein AP2. CHC, clathrin heavy chain CLC, clathrin light chain Nature Reviews Molecular Cell Biology 4, 409-414 (May 2003) 15  A cytoplasmic GTPase that is mainly involved in the scission of newly formed clathrin-coated vesicles from the membrane Scission Cytoplasm Plasma membrane 16 http://www.endocytosis.org/Endocytosis/CCVAssembly.html  The 2013 Nobel Prize in Physiology or Medicine Awarded jointly to James E. Rothman, Randy W. Schekman and Thomas C. Südhof for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells The three Nobel Laureates have discovered the molecular principles that govern how this cargo is delivered to the right place at the right time in the cell. The Nobel committee said: Schekman discovered a set of genes that were required for vesicle transport while Rothman revealed how proteins dock with their target membranes like two sides of a zipper Sudhof found out how vesicles release their cargo with precision. 17 Press Release 2013-10-07  Regulation of GPCR Signaling 1. Endocytosis 2. Desensitization and Resensitization, Downregulation and Upregulation 3. Recycling of GPCRs 18  Regulation of GPCR Signaling 1. Endocytosis 2. Desensitization and Resensitization, Downregulation and Upregulation 3. Recycling of GPCRs 19  Dynamin 20 Nature Reviews Neuroscience 2, 727-733 (October 2001)  Regulation of GPCR Signaling 1. Endocytosis 2. Desensitization and Resensitization, Downregulation and Upregulation 3. Recycling of GPCRs 22 Two major patterns:  Homologous Desensitization ◦ Incubation of an agonist with its receptor only attenuates the subsequent cellular response of this agonist through its receptor  Heterologous Desensitization ◦ Incubation of an agonist with a particular receptor attenuates the response to different other agonists working on different receptors within the cell ◦ It is a form of desensitization of one type of GPCR that can be induced by activation of another (heterologous) receptor 23  Desensitization Homologous Heterologous 1-Agonist-non-specific 1-Agonist-induced 2- Might result from activation of 2-Not influenced by activation of other receptors in the same cell other receptors in the same cell 3- Receptor not necessarily active Receptor is Phosphorylated by Receptor is Phosphorylated by 2nd messenger – dep kinases: GPCR kinases (GRKs) PKA or PKC β-arrestin mediated β-arrestin independent Receptor should be further p~ed by Usually followed by clathrin- GRKs to undergo subsequent mediated endocytosis of receptor clathrin –mediated endocytosis 24 Desensitization 25  Occurs only when the agonist is bound to the receptor (active receptor)  GRK phosphorylation of GPCR increases the affinity of the receptor to β-arrestin, a critical step that impairs coupling to G proteins  Binding of the β-arrestin to the phosphorylated receptor occurs only in cases of GRK-dependent phosphorylation 26  Phosphorylation of β2- adrenergic receptors by PKA diverts its coupling from Gs to Gi  The resulting activation of βγ subunits (partners of Gi) initiates a pathway of reactions leading to the activation of ERK and subsequent nuclear signals  However, desensitization of the newly coupled Gi signaling occurs through GRK-mediated phosphorylation 27 P~ at IC3 P~ at C-terminal 1 2 28  A negative feedback mechanism  PKA can efficiently phosphorylate inactive β2ARs  PKA activity can be stimulated by any receptor (including but not limited to β2AR) that promotes adenylyl cyclase activation. 29 Desensitization/Resensitization: (Studied on β2AR)  Desensitization: (Rapid) Within several minutes, the ability of receptors to mediate agonist-induced signaling diminishes  Resensitization: (Rapid) Agonist responsiveness of the receptor recovers quite rapidly. This recovery is thought to be important physiologically for mediating sustained (rather than transient) signaling responses in the continued presence of agonist. Rapid desensitization and resensitization of β2AR mediated signaling can occur without any detectable change in the total number of receptors present in cells or tissues 30 Handbook of Cell Signaling, 2nd edition Resensitization 31 Downregulation/Upregulation:  Downregulation:  Decrease in total number of receptor sites present in cells or tissues.  It generally occurs over a period of hours after prolonged agonist activation of receptors or activation of downstream signaling pathways  Functions to reduce cell or tissue responsiveness under conditions of prolonged agonist activation.  Upregulation:  Recovery of receptor number (and signaling responsiveness) after downregulation.  It also occurs relatively slowly, and typically requires biosynthesis of new receptor protein. The processes of downregulation and upregulation are thought to reflect primarily a change in the total number of GPCRs present in cells or tissues. 32 Handbook of Cell Signaling, 2nd edition  Desensitization/ Downregulation/ Resensitization Upregulation Rapid (within minutes) Slow (within hours) No effect on total number of GPCRs Reflects a change in total number of present GPCRs present Receptors are recycled Requires biosynthesis of new receptors Resensitization is important Downregulation functions to reduce physiologically for mediating cell or tissue responsiveness under sustained signaling responses conditions of prolonged agonist (rather than transient) activation. 33  Regulation of GPCR Signaling 1. Endocytosis 2. Desensitization and Resensitization Downregulation and Upregulation 3. Recycling of GPCRs 34 35 Determinants for GPCRs sorting are not completely understood, but may involve:  Factors that target the recycling of internalized GPCRs; these include: ◦ Dephosphorylation of internalized GPCRs inside endosomes by phosphatases. ◦ Transient interaction of GPCRs with β-arrestins ◦ The presence of recycling sequences in the C-terminus of a considerable number of GPCRs, which allows their interaction with recycling sorting proteins such as Na+/H+ exchanger regulatory factor1 (NHERF) and N-ethylmaleimide-sensitive factor (NSF)  Factors that target the degradation of internalized GPCRs; these include: ◦ Stable complex formation between GPCRs and β-arrestin mediated by:  the presence of phosphorylated S/T clusters in the C-terminus of GPCRs  ubiquitination of β-arrestin ◦ Ubiquitination of GPCRs, which allows for their recognition by the endosomal- sorting complex required for transport (ESCRT) machinery and targeting for degradation ◦ Other factors such as GPCR-associated sorting proteins (GASP) and sorting nexin-1 (SNX1) also play role in targeting for degradation 36  A. Role in Rapid Desensitization of GPCRs Explained earlier B. Role in Resensitization of GPCRs C. Role in Mediating Proteolytic Downregulation of GPCRs D. Role in Controlling the Specificity of Signal Transduction 37 Biasing the signaling downstream of βAR towards activation of ERK pathway: Generally involves the formation of a protein complex on endosome membranes. This protein complex includes, besides the internalized GPCRs, either: signal-transducing kinases (such as c-Src) recruited from the cytoplasm or receptor tyrosine kinases (such as EGFRs) co-endocytosed from the plasma membrane 38 Luttrell LM, Lefkowitz RJ. J Cell Sci. 2002;115:455–465.