Inhibition of p and 6 but Not K Opioid Binding to Membranes by Fab Fragments from a Monoclonal Antibody Directed against the Opioid Receptor*

Fab fragments from a monoclonal antibody, OR- 689.2.4, directed against the opioid receptor, selectively inhibited opioid binding to rat and guinea pig neural membranes. In a titratable manner, the Fab fragments noncompetitively inhibited the binding of the p selective peptide [~-Ala~,(Me)Phe~,Gly(0H)~][~H] enkephalin and the 6 selective peptide [~-Pen~,~-Pen'] r3H]enkepha1in (where Pen represents penicillamine) to neural membranes. In contrast, K opioid binding, as measured by the binding of [SH]bremazocine to rat neural membranes and guinea pig cerebellum in the presence of p and 6 blockers, was not significantly altered by the Fab fragments. In addition to blocking the binding of p and 6 ligands, the Fab fragments displaced bound opioids from the membranes. When p sites were blocked with [~-Ala~,(Me)Phe",Gly(OH)~]en-kephalin, the Fab fragments suppressed the binding of [~-Pen~,~-Pen'][~H]enkephalin to the same degree as when the p binding site was not blocked. The Fab fragments also inhibited binding to the p site regardless of whether or not the 6 site was blocked with [D-Pen2,D-Pen'lenkephalin. This monoclonal antibody is directed against a 35,000-dalton protein. Since the antibody is able to inhibit p and 6 binding but

the p site (1, 6, 7). @-Endorphin has almost equal affinity for the p and 6 binding sites (7, 8), though it may also bind to its own distinct opioid receptor, the 6 receptor (9, IO), and to a nonopioid receptor (11). The dynorphins have the highest affinity for the K opioid receptor (12, 13).
As a tool to probe the molecular basis of the multiple opioid receptors, a monoclonal antibody, OR-689.2.4, capable of partially inhibiting opioid binding to rat neural membranes, has been produced (14). This IgM was able to block and displace opioid ligands from rat neural membranes. The specificity of this immunoglobulin has been demonstrated by its inability to inhibit the binding of nonopioid ligands to neural membranes and the ineffectivenes of other mouse immunoglobulins to block the binding opioids to neural membranes (14). The antibody, directed against a 35,000-dalton protein, immunoprecipitated opioid binding sites from a solubilized preparation (14). Because it is an IgM with a molecular weight of 980,000, the ability of the antibody to penetrate tissue is hampered. As a consequence, a procedure was developed for obtaining Fab fragments, with a molecular weight of 48,000, from this IgM (15). This study describes the ability of OR-689.2.4 Fab fragments to selectively inhibit the different types of opioid binding sites in neural membranes. to rat neural membranes by 28% with a 15-min preincubation at 25 "C, by 35% with a 60-min preincubation a t 25 "C, and by 52% with a 24-h preincubation at 4 "C. By increasing the concentration of Fab fragments and the time of preincubation, it was possible to achieve an 80% inhibition in the binding of [3H]DAG0 to neural membranes.
The OR-689.2.4 Fab fragments also inhibited the binding of 3 nM [3H]DPDPE to rat neural membranes, as shown in Fig. 2. As with p peptide, the longer the preincubation of the membranes with the Fab fragments the greater the inhibition obtained with a certain concentration of Fab fragment. A concentration of 400 nM Fab fragments resulted in a 50% inhibition of the binding of 3 nM 13H]DPDPE, when a 60min preincubation at 25 "C was used. However with a 24-h preincubation at 4 "C, only 60 nM of Fab fragments was necessary to achieve a 50% inhibition in the binding of 3 nM [3H]DPDPE. Complete inhibition of the binding of [3H] DPDPE was obtained with 1 p M of OR-689.2.4 Fab fragments and a 24-h preincubation at 4 "C. Under identical conditions, the Fab fragments were able to inhibit the binding of the 6 ligand slightly better than the p ligand.
The ability of OR-689.2.4 Fab fragments to inhibit K binding to rat neural membranes, which have less than 20% K binding sites (20), and guinea pig cerebellum, which has been shown to contain 85% K opioid receptors (21) Most of the binding of [3H]bremazocine to rat neural membranes is to p and 6 sites, due to the fact that K sites account for less than 20% of the opioid binding sites. In the absence of p and 6 blockers, the Fab fragments inhibited the binding of 0.1 nM [3H]bremazocine to rat neural membranes by 27%, indicating that the Fab fragments could inhibit [3H]bremazocine binding to p and 6 sites. Table I1 shows the effect of the OR-689.2.4 Fab fragments on the binding of 0.1 nM [3H] bremazocine to guinea pig cerebellar membranes. The Fab fragments did not significantly suppress the binding of 0.1 nM [3H]bremazocine to guinea pig cerebellum, regardless of whether /I and 6 blockers were present. As previously reported (21) and confirmed in these studies, K binding sites account for 84-88% of the opioid binding sites in the guinea pig cerebellum.
To determine that the lack of inhibition of [3H]bremazocine binding to guinea pig cerebellum by the Fab fragments was not the result of species differences in the molecular structure of the opioid receptor, we investigated the ability of the Fab fragments to inhibit the binding of 1 nM [3H]DAG0 and 3 nM 13H]DPDPE to guinea pig neural membranes. As can be seen in Fig. 3 . 7). After a 60-min incubation with the Fab fragments, 0.5 nM [3H]DAG0 was added. As detailed in Table 111, the Fab fragments were able to inhibit the binding of 0.5 nM [3H] DAGO to membranes to the same degree regardless of whether 6 sites were or were not blocked by DPDPE.
To examine the reverse situation, DAGO at a concentration of 100 nM was used to block p binding sites. The Fab fragments at a concentration of 250 nM displaced only 14% of bound [3H]DAG0 (see Fig. 6). After the preincubation with DAGO and the addition of the Fab fragments, 5 nM [3H] DPDPE was added to the membranes to determine if the Fab fragments could inhibit the binding of [3H]DPDPE when p sites were blocked with ligand. As depicted in Table IV, the Fab fragments at a concentration of 250 nM inhibited the binding of 5 nM [3H]DPDPE by 38-40% regardless of whether the p sites were blocked with DAGO. These results indicate that the binding of p or 6 opioids to membranes does not result in a conformational change of the receptor such that the Fab fragments become ineffective in inhibiting binding to the unblocked site.

DISCUSSION
Elucidating the molecular basis of the multiple opioid receptors has proven to be a difficult task. A monoclonal antibody to the opioid receptor is a tool that will help determine the biochemical basis of the opioid receptor. The monoclonal IgM OR-689.2.4 has been shown to be specific for the opioid receptor (14). Because of the large size of a mouse IgM, 980,000 daltons, the ability of this antibody to penetrate neural membranes is restricted. A procedure was developed for generating Fab fragments from an IgM, effectively reducing the active size of the antibody from a molecular weight of 980,000 to 48,000 (15). With the Fab fragments, inhibition of opioid binding to neural membranes of greater than 90% was obtained. With the whole IgM, maximal inhibition obtained was approximately 35%, probably a consequence of the IgM's inability to sufficiently penetrate membranes (15).
The longer the preincubation of membranes with the Fab fragments, the greater the inhibition obtained with a certain concentration of Fab fragments. This is because most immunoglobulins have a much slower association and dissociation rate with the antigenic site than most receptor ligands have with their receptor. As a consequence, a 60-min incubation of membranes with the Fab fragments is not sufficient time for equilibrium to be reached. Thus, the longer the preincubation, the further toward equilibrium is the receptor-antibody complex. The Fab fragments inhibited opioid binding to p and 6 sites but not K sites. The Fab fragments inhibited the binding of [3H]DPDPE slightly better than [3H]DAG0. Whether this finding would be true for all p and 6 ligands is not known.
Due to the fact that guinea pig cerebellum is a rich source of K sites, this tissue was chosen in addition to rat neural membranes to investigate whether the Fab fragments could inhibit opioid binding to K sites. The Fab fragments did not suppress the binding of [3H]bremazocine to K sites in either rat or guinea pig membranes. Bremazocine has the highest affinity for K opioid binding sites, but also binds to p and 6 sites (7). In rat neural membranes, where a significant proportion of the binding of 0. to guinea pig neural membranes, indicating that the Fab fragments recognized a component of guinea pig p and 6 opioid receptor, but not the K receptor (Fig. 3). The antigenic site on the 35,000-dalton protein that the antibody is directed against is present in guinea pig as well as rat neural membranes.
The enkephalins and @-endorphin bind to p and 6 sites, but not significantly to K sites (1,(6)(7)(8). Distinguishing between p and 6 opioid binding sites has always been most difficult. Many opioids can differentiate between K sites and p- 6 sites, but not between p and 6 sites (7). A common high-affinity binding site, pl, has been proposed for opioids and enkephalins (22). A physical separation by sucrose gradient centrifugation of solubilized p and 6 receptors from K receptors has been obtained (23). The studies presented here suggest that there is a common component, a 35,000-dalton protein, shared by p and 6 opioid receptors but not the K receptor.
The OR-689.2.4 Fab fragments are noncompetitive inhibitors of p and 6 binding to neural membranes. The antibody is acting at a site distinct from the ligand binding site. The Fab fragments appear to be exerting an effect similar to the inhibition of agonist binding seen with sodium (24). The antibody will, however, inhibit antagonist binding to neural membranes (14). The Fab fragments must be altering the receptor conformation in such a manner as to render it unable to bind opioids. A stimulation in the binding of nerve growth factor to the nerve growth factor receptor on PC12 cells has been observed when PC12 cells were incubated with a monoclonal antibody directed against this receptor (25). Monoclonal antibodies directed against different epitopes on a receptor can influence ligand binding to the receptor. The OR-689.2.4 antibody appears to be directed against a three-dimensional epitope of the 35,000-dalton protein, concluded from the fact that sodium dodecyl sulfate will destroy the antigenic site (14). When 6 sites were blocked with DPDPE, the Fab fragments were able to inhibit the binding of [3H]DAG0 to the same degree as when 6 sites were not blocked with ligand. Under the conditions used, the binding of DPDPE to the 6 site did not change the conformation of the receptor in such a manner as to render it unable to bind [3H]DAG0. The same results were obtained when the p site was blocked, and binding to the 6 site was measured. It has previously been suggested that p and 6 opioid receptors may be interconvertible (26). While the studies presented here do not rule out this possibility, the binding of DAGO to the p site did not alter the receptor in a manner such that the Fab fragments could not block binding to the 6 site, and vice versa.
In summary, Fab fragments from the OR-689.2.4 IgM recognize a component common to both p and 6 opioid receptors in rat and guinea pig neural membranes but not K opioid receptors. This immunoglobulin should prove useful in the elucidation of the multiple opioid receptors.