Arrestin Is Required for Agonist-induced Trafficking of Voltage-dependent Calcium Channels

We have investigated the heterodimerization of ORL1 receptors and classical members of the opioid receptor family. All three classes of opioid receptors could be co-immunoprecipitated with ORL1 receptors from both transfected tsA-201 cell lysate and rat dorsal root ganglia lysate, suggesting that these receptors can form heterodimers. Consistent with this hypothesis, in cells expressing either one of the opioid receptors together with ORL1, prolonged ORL1 receptor activation via nociceptin application resulted in internalization of the opioid receptors. Conversely, -, ␦-, and -opioid receptor activation with the appropriate ligands triggered the internalization of ORL1. The -opioid receptor/ORL1 receptor heterodimers were shown to associate with N-type calcium channels, with activation of -opioid receptors triggering N-type channel internalization, but only in the presence of ORL1. Furthermore, the formation of opioid receptor/ORL1 receptor heterodimers attenuated the ORL1 receptor-mediated inhibition of N-type channels, in part because of constitutive opioid receptor activity. Collectively, our data support the existence of heterodimers between ORL1 and classical opioid receptors, with profound implications for effectors such as N-type calcium channels.ORL1 (opioid receptor-like 1) receptors (also known as NOP or nociceptin receptors) belong to the class of G␣ i/o -linked seven-helix transmembrane receptors (1, 2). They are structurally related to members of the classical opioid receptor family (i.e. -, ␦-, and -opioid receptors) but do not interact with known opioid receptor agonists or antagonists. Instead, they are activated by the endogenous ligand orphanin FQ (also known as nociceptin), a 17-amino acid polypeptide (3). ORL1 receptors are expressed in both the central and peripheral nervous systems; the physiological effects of receptor activation include stimulation of food intake, reduced anxiety, reduced withdrawal symptoms, and when activated peripherally, analgesia (4 -6). There is a functional cross-talk between ORL1 receptors and -opioid receptors such that chronic administration of the -receptor agonist morphine results in increased ORL1 receptor expression levels (7), whereas knock-out of the ORL1 receptor gene results in decreased morphine tolerance (8) without compensatory changes in opioid receptor expression (9). This may hint at the possibility of overlapping mechanisms controlling cellular expression levels of these two receptor subtypes.We have recently shown that ORL1 receptors physically interact with N-type calcium channels and that this interaction results in two distinct consequences: first, an agonist-independent inhibition of N-type channels due to constitutive receptor activity (10), and second, receptor-mediated trafficking of N-type channels to and from the plasma membrane (11). Neither of these phenomena appeared to occur with -opioid receptors (11). ORL1 receptors have also been shown to heterodimerize with -opioid receptors (12), with dimerized receptors showing altered sensitiv...

Section: Discussionmentioning

confidence: 99%

Heterodimerization of ORL1 and Opioid Receptors and Its Consequences for N-type Calcium Channel Regulation

Evans

You

Hameed

et al. 2010

“…However, with the methods used, we cannot exclude the possibility of very fast agonist-induced endocytosis of GABA B receptors coupled with rapid recycling back to the cell membrane. In this respect, it is interesting to note that a recent study analyzing GABA B receptor-induced internalization of calcium channels in cultured chick sensory neurons suggests the fast endocytosis of a complex of calcium channels, arrestin, and GABA B receptor 20 s after baclofen stimulation (33). Intracellular co-localization of calcium channels and arrestin with GABA B receptors strongly diminished already after 1 min, indicating a rapid and transient phenomenon.…”

Section: Discussionmentioning

confidence: 99%

γ-Aminobutyric Acid Type B Receptors Are Constitutively Internalized via the Clathrin-dependent Pathway and Targeted to Lysosomes for Degradation

Grampp

Sauter²,

Marković

et al. 2007

Receptor internalization is recognized as an important mechanism for rapidly regulating cell surface numbers of receptors. However, there are conflicting results on the existence of rapid endocytosis of ␥-aminobutyric acid, type B (GABA B ) receptors. Therefore, we analyzed internalization of GABA B receptors expressed in HEK 293 cells qualitatively and quantitatively using immunocytochemical, cell surface enzyme-linked immunosorbent assay, and biotinylation methods. The data indicate the existence of rapid constitutive receptor internalization, with the first endocytosed receptors being observed in proximity of the plasma membrane after 10 min. After 120 min, a loss of about 40 -50% of cell surface receptors was detected. Stimulation of GABA B receptors with GABA or baclofen did not enhance endocytosis of receptors, indicating the lack of agonistinduced internalization. The data suggest that GABA B receptors were endocytosed via the classical dynamin-and clathrin-dependent pathway and accumulated in an endosomal sorting compartment before being targeted to lysosomes for degradation. No evidence for recycling of receptors back to the cell surface was found. In conclusion, the results indicate the presence of constitutive internalization of GABA B receptors via clathrincoated pits, which resulted in lysosomal degradation of the receptors. GABA B3 receptors are G protein-coupled receptors that play an important role in the control of neurotransmission. They are widely expressed in the nervous system and have been implicated as potential targets for neurological diseases, such as epilepsy, pain, spasticity, addiction, schizophrenia, depression, and anxiety (for a review, see Ref. 1). GABA B receptors mediate slow inhibitory neurotransmission by either activating postsynaptically K ϩ channels or inhibiting presynaptically the release of neurotransmitters by modulation of Ca 2ϩ channels. On the structural level, functional GABA B receptors require the heterodimerization of two distinct seven-transmembrane proteins, termed GABA B1 and GABA B2 (2-7). Two main variants of GABA B1 have been reported (GABA B1a and GABA B1b (8)), which are generated by alternative promoter usage (9) and differ solely in their N-terminal domain. Heterodimerization of GABA B1a or GABA B1b with GABA B2 leads to two main GABA B receptor subtypes, GABA B1a /GABA B2 and GABA B1b /GABA B2 , which are abundantly expressed in all major brain structures (10 -13).An important aspect in the regulation of G protein-coupled receptors is their internalization or endocytosis. To protect cells against receptor overstimulation, the vast majority of G protein-coupled receptors desensitize upon prolonged agonist exposure, followed by rapid internalization. Many G protein-coupled receptors undergo phosphorylation upon agonist exposure by a G protein receptor kinase and subsequently recruit an arrestin protein (14). Arrestins often enhance phosphorylation, sterically interfere with binding of the G protein, and function as a signal for receptor endocytosis (15)...

“…We have previously used this peptide to interfere with ␤-Arr1-channel interaction in dorsal root ganglion neurons and block agonistinduced VDCC internalization (15). This peptide works as a "sponge" to bind the endogenous ␤-Arr1, which should result in the blockade of responses mediated by this molecule.…”

Section: ␤-Arr1 and Tyrosine Kinase Activity Are Required For Agonistmentioning

confidence: 99%

“…To test whether Ca v 1.2-␤-Arr1 interaction is required for ISO-induced internalization of VDCCs, we used cell-permeant peptides based on the sequence of the ␤-Arr1-binding site in Ca v 2.2 channel (Gallus gallus CDB1, aa 894 -944) (15). We have previously used this peptide to interfere with ␤-Arr1-channel interaction in dorsal root ganglion neurons and block agonistinduced VDCC internalization (15).…”

Section: ␤-Arr1 and Tyrosine Kinase Activity Are Required For Agonistmentioning

confidence: 99%

β-Adrenergic Receptor Activation Induces Internalization of Cardiac Cav1.2 Channel Complexes through a β-Arrestin 1-mediated Pathway

Lipsky

Potts

Tarzami

et al. 2008

Self Cite

Voltage-dependent calcium channels (VDCCs) play a pivotal role in normal excitation-contraction coupling in cardiac myocytes. These channels can be modulated through activation of ␤-adrenergic receptors (␤-ARs), which leads to an increase in calcium current (I Ca-L ) density through cardiac Ca v 1 channels as a result of phosphorylation by cAMP-dependent protein kinase A. Changes in I Ca-L density and kinetics in heart failure often occur in the absence of changes in Ca v 1 channel expression, arguing for the importance of posttranslational modification of these channels in heart disease. The precise molecular mechanisms that govern the regulation of VDCCs and their cell surface localization remain unknown. Our data show that sustained ␤-AR activation induces internalization of a cardiac macromolecular complex involving VDCC and ␤-arrestin 1 (␤-Arr1) into clathrincoated vesicles. Pretreatment of myocytes with pertussis toxin prevents the internalization of VDCCs, suggesting that G i/o mediates this response. A peptide that selectively disrupts the interaction between Ca V 1.2 and ␤-Arr1 and tyrosine kinase inhibitors readily prevent agonist-induced VDCC internalization. These observations suggest that VDCC trafficking is mediated by G protein switching to G i of the ␤-AR, which plays a prominent role in various cardiac pathologies associated with a hyperadrenergic state, such as hypertrophy and heart failure. Regulation of voltage-dependent calcium channels (VDCCs)3 plays a pivotal role in excitation-contraction coupling in cardiac myocytes. During the action potential upstroke, membrane depolarization causes the opening of VDCCs, encoded by the pore-forming ␣ 1 subunit, Ca v 1.2 (1). Ca 2ϩ entry through VDCCs triggers the release of Ca 2ϩ from the sarcoplasmic reticulum via ryanodine receptors. Although the regulation of VDCCs in the heart has been extensively studied, key molecular mechanisms underlying channel function, trafficking, membrane targeting, retention, and internalization remain unknown. Activation of the ␤-AR, a G protein-coupled receptor (GPCR), leads to positive inotropic effects mediated by phosphorylation of the VDCC via cAMP-dependent protein kinase A (2). This, however, is a transient phenomenon since persistent activation of the receptor causes its subsequent phosphorylation by GPCR kinases (GRKs) (3), causing the ␤-AR to become a target for arrestin (4), which mediates the recruitment of the receptor into clathrin-coated vesicles (5).In addition to decreasing single channel permeability, persistent membrane depolarization can regulate the number of Ca v 1.2 channels at the plasma membrane. For example, sustained KCl-induced depolarization of rat cortical neurons effectively decreases Ca v 1.2 channel activity (6). Ca v 1.2 channels have been proposed to contain a membrane-targeting domain within their calmodulin (CaM)-binding domain in the C terminus (7). Pitt and colleagues (8) showed that Ca 2ϩ -CaM interaction with this domain accelerated the rate of trafficking of Ca v 1.2 channels to ...