Seven-transmembrane receptors and ubiquitination

Abstract

Regulation of protein function by posttranslational modification plays an important role in many biological pathways. The most well known among such modifications is protein phosphorylation performed by highly specific protein kinases. In the past decade, however, covalent linkage of the low-molecular-weight protein ubiquitin to substrate proteins (protein ubiquitination) has proven to be yet another widely used mechanism of protein regulation playing a crucial role in virtually all aspects of cellular functions. This review highlights some of the recently discovered and provocative roles for ubiquitination in the regulation of the life cycle and signal transduction properties of 7-transmembrane receptors that serve to integrate many biological functions and play fundamental roles in cardiovascular homeostasis.

Figure 1. 7TMR trafficking and Ubiquitination

A. General scheme of receptor trafficking. Stimulation of cell surface 7TMRs leads to G protein dependent signaling (not shown) as well as G protein coupled receptor kinase mediated receptor phosphorylation followed by recruitment of β-arrestin. Subsequently, either receptors or receptor-β-arrestin complexes (not shown) internalize into early endosomes via clathrin-coated vesicles (CCV). From here receptors traffic to late endosomes and then to lysosomes where they are degraded. Alternatively some receptors take a recycling path back to the plasma membrane. B. The process of ubiquitination: The C-terminal glycine residue of ubiquitin is activated by the enzyme E1 (ubiquitin activating enzyme) in an ATP-dependent reaction resulting in an intermediary ubiquitin adenylate, release of PPi, followed by the attachment of ubiquitin to a cysteine residue in E1. Activated ubiquitin is next transferred to an active cysteine in E2 (ubiquitin carrier enzyme). In the third step, a ubiquitin protein ligase E3 links the carboxyl terminus of ubiquitin to an ε-amino group of a lysine residue in the substrate protein. Attachment of a single ubiquitin at a single site is called monoubiquitination, whereas one ubiquitin at multiple sites is called multi-monoubiquitination. When subsequent additions of ubiquitin are made to a lysine within the previously added ubiquitin, a polyubiquitin chain is formed. C. The yeast 7TMR Ste2 is monoubiquitinated in response to pheromone alpha factor. This monoubiquitination is required for both internalization and subsequent degradation of the receptor in the yeast vacuole. Ste2 receptors which are not ubiquitinated in response to ligand are retained at the cell surface. D. Mammalian 7TMRs are either polyubiquitinated {e.g. β2 adrenergic receptor (β2AR); V2 vasopressin receptor (V2R); Neurokinin-1 receptor (NK-1R)} or monoubiquitinated {e.g. chemokine receptor CXCR4; protease activated receptor 2 (PAR2), platelet activating factor receptor (PAFR)} in response to agonist induction. For the above receptors (except PAFR), mutagenesis of ubiquitination attachment sites (lysine residues) abolishes ubiquitination and receptor degradation in lysosomes, but does not affect receptor internalization per se. Receptors which are not ubiquitinated undergo endocytosis, but they are not sorted to lysosomes. β-arrestin is recruited (indicated) and ubiquitinated (not shown) irrespective of the status of receptor ubiquitination.

Figure 1. 7TMR trafficking and Ubiquitination

A. General scheme of receptor trafficking. Stimulation of cell surface 7TMRs leads to G protein dependent signaling (not shown) as well as G protein coupled receptor kinase mediated receptor phosphorylation followed by recruitment of β-arrestin. Subsequently, either receptors or receptor-β-arrestin complexes (not shown) internalize into early endosomes via clathrin-coated vesicles (CCV). From here receptors traffic to late endosomes and then to lysosomes where they are degraded. Alternatively some receptors take a recycling path back to the plasma membrane. B. The process of ubiquitination: The C-terminal glycine residue of ubiquitin is activated by the enzyme E1 (ubiquitin activating enzyme) in an ATP-dependent reaction resulting in an intermediary ubiquitin adenylate, release of PPi, followed by the attachment of ubiquitin to a cysteine residue in E1. Activated ubiquitin is next transferred to an active cysteine in E2 (ubiquitin carrier enzyme). In the third step, a ubiquitin protein ligase E3 links the carboxyl terminus of ubiquitin to an ε-amino group of a lysine residue in the substrate protein. Attachment of a single ubiquitin at a single site is called monoubiquitination, whereas one ubiquitin at multiple sites is called multi-monoubiquitination. When subsequent additions of ubiquitin are made to a lysine within the previously added ubiquitin, a polyubiquitin chain is formed. C. The yeast 7TMR Ste2 is monoubiquitinated in response to pheromone alpha factor. This monoubiquitination is required for both internalization and subsequent degradation of the receptor in the yeast vacuole. Ste2 receptors which are not ubiquitinated in response to ligand are retained at the cell surface. D. Mammalian 7TMRs are either polyubiquitinated {e.g. β2 adrenergic receptor (β2AR); V2 vasopressin receptor (V2R); Neurokinin-1 receptor (NK-1R)} or monoubiquitinated {e.g. chemokine receptor CXCR4; protease activated receptor 2 (PAR2), platelet activating factor receptor (PAFR)} in response to agonist induction. For the above receptors (except PAFR), mutagenesis of ubiquitination attachment sites (lysine residues) abolishes ubiquitination and receptor degradation in lysosomes, but does not affect receptor internalization per se. Receptors which are not ubiquitinated undergo endocytosis, but they are not sorted to lysosomes. β-arrestin is recruited (indicated) and ubiquitinated (not shown) irrespective of the status of receptor ubiquitination.

Figure 1. 7TMR trafficking and Ubiquitination

A. General scheme of receptor trafficking. Stimulation of cell surface 7TMRs leads to G protein dependent signaling (not shown) as well as G protein coupled receptor kinase mediated receptor phosphorylation followed by recruitment of β-arrestin. Subsequently, either receptors or receptor-β-arrestin complexes (not shown) internalize into early endosomes via clathrin-coated vesicles (CCV). From here receptors traffic to late endosomes and then to lysosomes where they are degraded. Alternatively some receptors take a recycling path back to the plasma membrane. B. The process of ubiquitination: The C-terminal glycine residue of ubiquitin is activated by the enzyme E1 (ubiquitin activating enzyme) in an ATP-dependent reaction resulting in an intermediary ubiquitin adenylate, release of PPi, followed by the attachment of ubiquitin to a cysteine residue in E1. Activated ubiquitin is next transferred to an active cysteine in E2 (ubiquitin carrier enzyme). In the third step, a ubiquitin protein ligase E3 links the carboxyl terminus of ubiquitin to an ε-amino group of a lysine residue in the substrate protein. Attachment of a single ubiquitin at a single site is called monoubiquitination, whereas one ubiquitin at multiple sites is called multi-monoubiquitination. When subsequent additions of ubiquitin are made to a lysine within the previously added ubiquitin, a polyubiquitin chain is formed. C. The yeast 7TMR Ste2 is monoubiquitinated in response to pheromone alpha factor. This monoubiquitination is required for both internalization and subsequent degradation of the receptor in the yeast vacuole. Ste2 receptors which are not ubiquitinated in response to ligand are retained at the cell surface. D. Mammalian 7TMRs are either polyubiquitinated {e.g. β2 adrenergic receptor (β2AR); V2 vasopressin receptor (V2R); Neurokinin-1 receptor (NK-1R)} or monoubiquitinated {e.g. chemokine receptor CXCR4; protease activated receptor 2 (PAR2), platelet activating factor receptor (PAFR)} in response to agonist induction. For the above receptors (except PAFR), mutagenesis of ubiquitination attachment sites (lysine residues) abolishes ubiquitination and receptor degradation in lysosomes, but does not affect receptor internalization per se. Receptors which are not ubiquitinated undergo endocytosis, but they are not sorted to lysosomes. β-arrestin is recruited (indicated) and ubiquitinated (not shown) irrespective of the status of receptor ubiquitination.

Figure 2. 7TMR regulatory pathways and proteasomal degradation

1. The proteasomal degradative pathway and ER quality control mechanisms are closely coupled. When misfolded upon synthesis, 7TMRs such as the delta opioid receptor (δOR), calcium sensing receptor (CaR) and thyrotropin releasing hormone receptor (TRHR) are ubiquitinated, retrotranslocated in a Sec61 dependent manner and degraded by the 26S proteasomal complex. 2. The metabotropic glutamate receptor (mGluR5) and Follitropin receptor (FSHR) are reported to be ubiquitinated and degraded by the proteasomal pathway. 3. Agonist stimulation of the β2AR leads to rapid polyubiquitination of G protein-coupled receptor kinase 2 (GRK2). This GRK2 regulation involves β-arrestin and Mdm2-dependent ubiquitination (not shown). Polyubiquitination of the Gαs subunit is also induced upon activation of the β2AR. Whether 7TMR stimulation plays a role in other examples of G protein subunit ubiquitination is currently unknown. These examples are discussed in the text. 4. Stimulation of Gαq-coupled receptors (e.g. muscarinic acetylcholine receptors) leads to the polyubiquitination and proteasome dependent degradation of Ins(1,4,5)P₃ receptors located in the ER membrane resulting in a suppression of Ins(1,4,5)P₃ induced Ca²⁺ mobilization. 5. Recent studies indicate that USP4, a deubiquitinating enzyme, can remove the polyubiquitin chains on ubiquitinated A2A adenosine receptors, thereby preventing receptor degradation and promoting 7TMR expression on the cell surface.

Figure 3. β-arrestin’s role in endocytosis, signaling and receptor ubiquitination

A. Patterns of β-arrestin ubiquitination correlate with the stability and localization of 7TMR signalosomes. Agonist–stimulation of 7TMRs leads to phosphorylation of serine-threonine residues in the cytoplasmic domains of the receptor by G protein-coupled receptor kinases. Phosphorylated receptors recruit the cytosolic adaptor protein, β-arrestin. β-arrestin binding interdicts further G protein coupling, leading to the ‘desensitization’ of second-messenger initiated pathways. β-arrestin acts as both a clathrin and AP2 adaptor and facilitates receptor internalization via clathrin coated vesicles. Most 7TMRs are capable of recruiting β-arrestin. However, the nature of β-arrestin-7TMR complexes can be transient (class A receptors) or stable (class B receptors). Notably, the ubiquitination patterns of β-arrestin correlates with the above binding patterns. Activation of class A receptors induces transient ubiquitination; β-arrestins are rapidly deubiquitinated and dissociate from the internalizing receptor. Activation of class B receptors induces sustained β-arrestin ubiquitination and the formation of stable endocytic complexes. While an ERK signaling scaffold is formed at the plasma membrane upon stimulation of either receptor type, only in the case of class B receptors are these signals further seen localized to perinuclear endosomal compartments representing sustained ERK phosphorylation. B. β-arrestin functions as an E3 ubiquitin ligase adaptor. For the 7TMRs β2AR and V2R, modification by ubiquitin requires the cellular expression of the β-arrestin2 isoform. The E3 ligase(s) involved remain to be determined. The insulin-like growth factor-1 receptor, recruits both β-arrestin 1 and 2. Polyubiquitination and proteasomal degradation of this receptor was recently shown to require both β-arrestin1 and Mdm2 activity. Kurtz, the single nonvisual arrestin expressed in Drosophila, plays a crucial and synergistic role in Deltex-mediated Notch ubiquitination and degradation.

Figure 3. β-arrestin’s role in endocytosis, signaling and receptor ubiquitination

A. Patterns of β-arrestin ubiquitination correlate with the stability and localization of 7TMR signalosomes. Agonist–stimulation of 7TMRs leads to phosphorylation of serine-threonine residues in the cytoplasmic domains of the receptor by G protein-coupled receptor kinases. Phosphorylated receptors recruit the cytosolic adaptor protein, β-arrestin. β-arrestin binding interdicts further G protein coupling, leading to the ‘desensitization’ of second-messenger initiated pathways. β-arrestin acts as both a clathrin and AP2 adaptor and facilitates receptor internalization via clathrin coated vesicles. Most 7TMRs are capable of recruiting β-arrestin. However, the nature of β-arrestin-7TMR complexes can be transient (class A receptors) or stable (class B receptors). Notably, the ubiquitination patterns of β-arrestin correlates with the above binding patterns. Activation of class A receptors induces transient ubiquitination; β-arrestins are rapidly deubiquitinated and dissociate from the internalizing receptor. Activation of class B receptors induces sustained β-arrestin ubiquitination and the formation of stable endocytic complexes. While an ERK signaling scaffold is formed at the plasma membrane upon stimulation of either receptor type, only in the case of class B receptors are these signals further seen localized to perinuclear endosomal compartments representing sustained ERK phosphorylation. B. β-arrestin functions as an E3 ubiquitin ligase adaptor. For the 7TMRs β2AR and V2R, modification by ubiquitin requires the cellular expression of the β-arrestin2 isoform. The E3 ligase(s) involved remain to be determined. The insulin-like growth factor-1 receptor, recruits both β-arrestin 1 and 2. Polyubiquitination and proteasomal degradation of this receptor was recently shown to require both β-arrestin1 and Mdm2 activity. Kurtz, the single nonvisual arrestin expressed in Drosophila, plays a crucial and synergistic role in Deltex-mediated Notch ubiquitination and degradation.