GPCRs undergo conformational changes following receptor activation. Although the involvement of the transmembrane regions in conformational changes following the binding of the agonist to the receptor has been extensively documented (1
), not much is known about the involvement of the N-terminal region. Antibodies have been useful tools in exploring the domains involved in activity-mediated conformational changes of signaling proteins, including receptors (19
). For example, a monoclonal antibody to the N-terminal region of rhodopsin exhibited a higher degree of recognition for bleached (activated) receptors than for unbleached (inactive) receptors even after detergent treatment. This indicates that photoactivation of rhodopsin induces a conformational change at the N terminus that exposes an epitope that is recognized by the monoclonal antibody (22
). In this study, we generated antibodies to distinct regions of the N terminus of μ
-opioid receptors to show that a region within the N terminus undergoes significant local movement following receptor activation. We find that this could be a general mechanism shared by other members of family A, since we observe that irrespective of the nature of the ligand or G-protein selectivity, receptor activation leads to the exposure of the epitope recognized by antibodies targeted to the midportion of the N terminus.
The role of the N-terminal region in ligand binding and receptor activation has been mainly explored in receptors with long N-terminal regions. For example, for glycoprotein hormone receptors, the long N-terminal tail constitutes the primary high affinity and selective binding site for receptor agonists (12
). In the case of receptors of family C, the very large extracellular N terminus is organized into a domain called the Venus flytrap module that contains the ligand binding pocket (37
). Interestingly, the smaller N-terminal tail of family A GPCRs, such as opioid receptors as well as other peptide or amine receptors, has also been proposed to participate in receptor activation (39
). In the case of δ
-opioid receptors, a random mutagenesis study identified 5 amino acids in the N-terminal region that enhanced the spontaneous activity of the receptor (15
). In this study, each mutation substantially modified the chemical nature of the amino acid side chain, suggesting that the N terminus of δ
receptors is folded as a domain whose structure and spatial orientation affects receptor function (15
). This is also suggested by the structure of rhodopsin, in which the N terminus is folded as a β
-sheet over the helical bundle covering it like a lid (40
). Although a comprehensive molecular mechanism for receptor activation that includes the extracellular loops and the N- and C-terminal tails is not yet available, the accumulating evidence to date is consistent with the notion that these regions of the receptor undergo substantial structural perturbations upon activation.
An observation in this study has been that increased recognition of the receptor by antibodies targeted to the midportion of the N-terminal region of μ-opioid receptors remains for a prolonged period (ever after the removal of the agonist). In addition, the formation of the ternary complex does not appear to be sufficient, since the recognition persists for a prolonged period of time (well beyond the formation of the ternary complex). It can be postulated that the antibody recognizes the activation-mediated post-translationally modified receptor. It should be pointed out that our assays (in whole cells and membranes) were carried out under conditions that do not allow receptor internalization (and dephosphorylation/resensitization). Hence, the changes in receptor conformation would persist for a long time, as seen in our studies with cells, membranes, and animals. Taken together, these results are consistent with the notion that the SA25 antibody recognizes changes (in the N terminus) that are induced by the activation-mediated long lasting changes to the receptor. We are currently exploring these possibilities.
Although a number of studies have investigated the activation state of heterogeneously expressed opioid receptors, a major research focus has been to identify brain regions and molecular partners playing critical roles in the development of side effects, such as tolerance to and physical dependence on opiates. However, no definite model has emerged that could be used to design a new category of drugs as powerful as morphine but with less abuse potential. This is partly due to difficulties in distinguishing the brain regions targeted by a drug, which depend on the route of administration, dose of the drug, and its bioavailability. For example, morphine has been shown to induce the activation of mitogen-activated protein kinase in a set of cells quite distinct from those that express μ
). Studies have also shown that the pharmacological properties of a ligand in vitro
can be different from those seen in vivo
. For example, [D-Pen2
]enkephalin and deltorphin II, two highly δ
-selective peptide agonists, are thought to induce analgesia through μ
). The lack of suitable reagents, thus far, has not allowed the direct evaluation of the extent of activation of receptors of interest. Attempts have been made to probe this using indirect markers (mitogen-activated protein kinase and GTPγ
S) or receptor knock-out mice. Apart from the radiolabeled GTPγ
S binding performed on slices (43
), few techniques are available that allow the investigation of the spatio-temporal dynamics of receptor activation. This emphasizes the dire need for reliable tools for identification of brain regions where GPCRs are activated at the cellular level. Our antibodies represent a useful and direct approach compared with other time-consuming and labor-intensive techniques. This approach seems to be applicable to many family A GPCRs and opens the way to examine the localization of active receptors as well as the extent of modulation of receptor activity by cross-talk between receptors.
The results of this study not only demonstrate the structural mobility exhibited by the small N-terminal region of distinct types of family A GPCRs but also provide a new and powerful technique to examine the duration and extent of activation of endogenous receptors as well as to screen for drugs that are allosteric modulators of family A GPCRs, which would be of potential therapeutic value.