Transmembrane Domains

16 May

TM residues within the lipophilic environment of the cell membrane are key in ligand recognition and/or signal transduction and are expected to be oriented toward a relatively hydrophilic central cavity (Surratt et al. 1994). Structural motives important for ligand binding and subtype specificity have been iden- tified for the opioid  receptors  from experimental  mutagenesis  studies  and computer modeling. A current view of how GPCRs achieve the transition  be- tween resting and active conformations  to convey external signals across the

cell membrane  is that ligands bind with hydrophilic  and aromatic  residues within the helical core. This triggers outward movements of TM helices 3, 6, and 7 to promote  the formation  of the active receptor state that results in G protein coupling and signal transduction, as it was shown for the structurally related muscarinic receptor (Hulme et al. 1999). Charged and polar amino acid residues are useful starting points in identifying important residues for ago- nist and antagonist binding within the binding pocket. For opioid receptors, histidine, asparagine, and tyrosine residues within TM 3, 6, and 7 are critical for receptor activation (Mansour et al. 1997). Random mutagenesis is another tool to identify important  structure–function relationships.  Such a strategy

with the entire δ-receptor revealed 30 point mutations leading to a constitutive activity of the receptor (Decaillot et al. 2003). Mutagenesis studies are useful tools to identify potential domains important for ligand binding. However, they

do not allow the definitive identification of binding sites, because the mutation of single amino acids could affect the amino acid side-chain interaction  and subsequently the secondary and/or tertiary structure of the entire receptor.

C-Terminal Tail

The C-terminal portion of the opioid receptor determines coupling to second messenger  molecules and  is important for receptor  trafficking. μ-receptor point  mutations  of any of the Ser/Thr within the C-terminal  tail result  in significant reductions in the rate of receptor internalization  in HEK cell lines (Koch et al. 1998, 2001). This was confirmed using CHO-K1 cells expressing μ-receptors  and a mutant  form, in which all Ser and Thr residues from the third  cytoplasmic loop and C-terminal  were changed to alanine (Capeyrou et al. 1997; Wang 2000).

Signal Transduction

Opioid receptors  are prototypical  Gi /Go-coupled  receptors.  Opioid signals are efficiently blocked by pertussis toxin that adenosine diphosphate  (ADP)-

Fig. 2a Opioid ligands induce a conformational change at the receptor which allows coupling of G proteins to the receptor. The heterotrimeric G protein dissociates into active Gα and Gβγ subunits (a) which can subsequently inhibit adenylyl cyclase (b), decrease the conductance of voltage-gated Ca2+  channels, open rectifying K+ channels (c), or activate the PLC/PKC pathway (d) that modulates Ca2+  channel activity in the plasma membrane  (e). b Opioid receptor desensitization and trafficking is activated by G protein-coupled receptor kinases (GRK). After arrestin binding, the receptor is in a desensitized state at the plasma membrane (a). Arrestin-bound receptors can then be internalized via a clathrin-dependent pathway, and either be recycled to the cell surface (b) or degraded in lysosomes (c)

ribosylates and inactivates the α-subunits of Gi/Go proteins. After opioid ago- nists bind to the receptor, dissociation of the trimeric G protein complex into Gα- and Gβγ-subunits can subsequently  lead to the inhibition  of cyclic 3 5 adenylyl cyclase (cAMP) and/or to direct interaction with K+, Ca2+, and other ion channels in the membrane  (Fig. 2a). Ion-channel  regulation  by opioids

is mainly mediated via direct G protein βγ-subunits (Herlitze et al. 1996). All three opioid receptors couple to various N, T-type and P/Q-type Ca2+ channels, suppress Ca2+ influx and subsequently the excitation and/or neurotransmitter release in many neuronal  systems. A prominent  example is the inhibition  of (pronociceptive)  substance P release from primary afferent sensory neurons in the spinal cord and from their peripheral  terminals  (Kondo et al. 2005).

At the postsynaptic  membrane,  opioid receptors  mediate hyperpolarization by activating K+  channels, thereby preventing  excitation or propagation  of action potentials.  Four families of potassium  channels play important roles in antinociception,  including voltage-gated K+  channels (Kv), inward recti-

fier K+  channels  (Kir), calcium-activated  K+  channels  (KCa), and two-pore K+ channels (K2P) (Ocana et al. 2004). In the central nervous system (CNS), opioid-induced  antinociception  is associated with activation of voltage-gated

K+ channels of the Kir channel family (Ocana et al. 1995). Some opioids also induce opening of calcium-activated K+ channels (Stretton et al. 1992).

Apart  from  Ca2+  and  K+  channels,  opioid  receptors  may  regulate  the

functions of other ion channels. For example, opioids suppress tetrodotoxin- resistant sodium-selective and nonselective cation currents, which are mainly expressed in nociceptors (Gold and Levine 1996; Ingram and Williams 1994). In addition, a number of studies indicate that opioids can directly modulate N - methyl-d-aspartate (NMDA) receptors at presynaptic and postsynaptic  sites within the CNS (Mao 1999). NMDA receptors  can be blocked at the same

site where Mg2+ and ketamine interfere with the ion channel. In addition, we recently showed that activation of opioid receptors  modulates  the transient

receptor  potential  vanilloid type 1 (TRPV1), a member  of the ligand gated ion channels (J. Endres-Becker, P.A. Heppenstall, S.A. Mousa, A. Oksche, M.

Schäfer, C. Stein, C. Zöllner, submitted  for publication).  TRPV1 is involved in thermosensation and nociception  and is mainly expressed in peripheral sensory neurons. It was also suggested that opioid receptors regulate the phos- pholipase Cβ (PLCβ) pathway via Gβγ-subunits (Chan et al. 1995) or Gq proteins (Rubovitch et al. 2003). PLC activation mobilizes phosphokinase C (PKC) that opens calcium channels in the plasma membrane. The entry of Ca2+ into the cell stimulates calcium-activated adenylyl cyclases to produce cAMP. However,

the physiological relevance of such a potential bidirectional regulation of in- tracellular cAMP by opioid receptors is not completely solved at the moment and most data indicate that opioids mainly inhibit cAMP production.

Mitogen-activated  protein  kinase  (MAPK), also known  as extracellular signal-regulated  kinase (ERK), is regarded  as a major  pathway for growth factor signaling from the cell surface to the nucleus (Seger and Krebs 1995). MAPK activation has been noted in response to agonist stimulation  of many GPCRs, including all opioid receptor types (Belcheva et al. 1998; Ignatova et al.

1999; Schulz et al. 2004a). A suggested mechanism includes the activation of PLC, generating diacylglycerol that binds to PKCε, leading to its phosphory- lation. PKCε can then signal to matrix metalloproteinases,  which can cleave

membrane-anchored epidermal  growth factor receptor  (EGF)-type ligands, thereby initiating  EGF receptor  transactivation and ultimately activation of the MAPK phosphorylation cascade (Belcheva et al. 2005; Pierce et al. 2001). So far, little is known about the functional significance of this phenomenon. Besides an impact of opioids on neural development, the MAPK pathway might be involved in homologous desensitization of the μ-receptor (Polakiewicz et al.

1998; Schmidt et al. 2000). Future studies might investigate the role of the MAPK

cascade in opioid tolerance and dependence.


Opioid Tolerance


In Vitro

On the cellular level long-term  opioid treatment  can result in the eventual loss of opioid receptor-activated function  (i.e., desensitization).  Three gen- eral mechanisms are associated with desensitization  of GPCRs: (1) receptor phosphorylation, (2) receptor  internalization  and/or  sequestration,  and (3) receptor  downregulation  (i.e., a reduced total number  of receptors).  Opioid receptors  are substrates  for second  messenger  kinases  (i.e., PKC) and  for members  of the GPCR kinases (GRK). Opioid receptor  phosphorylation by these kinases increases the affinity to arrestin  molecules. Arrestin-receptor complexes sterically prevent coupling between receptor  and G proteins  and promote  internalization  via clathrin-dependent pathways (Fig. 2b; Law et al.

2000). Agonist-induced internalization  of the receptor via the endocytic path- way has been thought  to contribute  directly to tolerance by decreasing the number  of opioid receptors on the cell surface. However, more recent stud- ies have shown that  morphine  fails to promote  endocytosis  of opioid  re- ceptors  in cultured  cells (Eisinger et al. 2002) and  native neurons  (Stern- ini et al. 1996), although  it is highly efficient in inducing  tolerance  in vivo (Sim et al. 1996). Moreover, endocytosis and recycling of the opioid recep- tor  was shown  to  dramatically  decrease  opioid  tolerance  and  withdrawal (Koch et al. 2005). These findings suggest that desensitization  and receptor internalization  might be a protective  mechanism  and prevent  the develop- ment of tolerance. Drugs that do not cause receptor internalization,  such as morphine,  may have higher propensities  to induce tolerance. In contrast  to full agonists such as [D-Ala(2)-MePhe(4)-Gly-ol]enkephalin (DAMGO), mor- phine stimulated a selective phosphorylation of the carboxy-terminal residue

375, indicating that morphine-desensitized receptors remained at the plasma membrane in a Serin-375-phosphorylated state for prolonged periods (Schulz et al. 2004b). Recycling of opioid  receptors  to the plasma  membrane  pro-

motes rapid resensitization of signal transduction, whereas targeting to lyso- somes leads to proteolytic  downregulation.  A protein  that  binds  preferen-

tially to the cytoplasmic tail of opioid receptors was identified and named as GPCR-associated sorting protein  (GASP) (Whistler et al. 2002). It was sug- gested that GASPs modulate lysosomal sorting and functional downregulation of GPCRs.


In Vivo

Tolerance in vivo describes the phenomenon  that the magnitude  of a given opioid effect decreases with repeated administration of the same opioid dose, or that increasing doses of an opioid are needed to produce the same effect. All opioid  effects (e.g., analgesia, respiratory  depression,  sedation,  consti- pation)  can be subject to tolerance  development.  It is evident that  opiate- induced  adaptations  occur at multiple  levels in the nervous  and other  or- gan systems, beginning with regulation  of opioid receptors  themselves and extending to complex networks including learned behavior and genetic fac- tors (von Zastrow 2004). The importance  of opioid receptor endocytosis for chronic adaptation  of the intact nervous system is not yet understood.  Indi- rect evidence that arrestin-dependent μ-receptor desensitization  contributes to morphine  tolerance  in vivo comes from studies in arrestin-3  and GRK3 knockout mice. Functional deletion of the arrestin-3 gene resulted in remark- able potentiation and prolongation  of the analgesic effect of morphine (Bohn et al. 2000; Bohn et al. 2002) but had no effect on the acute antinociceptive potency of etorphine,  fentanyl, or methadone  (Bohn et al. 2004). However, to date the majority of studies on opioid tolerance have been performed  in the absence of painful tissue injury. This may explain some of the discrep- ancies between experimental  (Smith et al. 2003) and clinical studies (Stein et al. 1996). Tolerance is not ubiquitously observed in clinical routine and is in many cases explained by increasing nociceptive stimulation with progressing disease (e.g., cancer pain; Zech et al. 1995). Animal models of pathological situations  have also produced  evidence for a reversal of tolerance  to mor- phine, e.g., during intestinal inflammation (Pol and Puig 1997). These findings indicate differences in the development of opioid tolerance (and possibly in receptor desensitization and recycling) under pathological situations. Future studies are necessary on the development of opioid receptor tolerance in the presence of postoperative pain, arthritis, or other types of inflammatory  and chronic pain.

Several investigators  have focused on the concept  that  tolerance  can be counteracted  by NMDA receptor  antagonists  (Mao 1999; Price et al. 2000). NMDA receptors are a subclass of excitatory amino acid receptors that, once activated, facilitate calcium influx into neurons. Although it has been shown NMDA receptor  antagonists,  e.g., MK801, inhibit  tolerance  to the analgesic effects of repeated  morphine  administration (Elliott et al. 1995; Price et al.

2000), the underlying mechanisms have not been fully elucidated.

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