Guanine Nucleotide Regulation of B2 Kinin Receptors TIME-DEPENDENT

the sensi-

Low affinity binding exhibited relatively rapid rates of association (12,bs = 1.40 X lo-' s-') and dissociation (kel = 3.82 x 10e3 s-l), while high affinity binding exhibited considerably slower rates (Iz,b = 9.52 x 10m4 s-l and k-1 = 4.43 x lo-' s-l). Pre-equilibrium dissociation kinetics revealed that formation of high affinity binding was characterized as a time-dependent accumulation of the slow dissociation rate at the expense of at least one other more rapid dissociation rate. In the presence of 10 pM guanyl-5'-yl imidodiphosphate (Gpp(NH)p), at least two binding components were resolved with KD values of 37 pM (12%) and 444 PM (88%). Gpp(NH)p apparently specifically perturbed high affinity binding by completely preventing the accumulation of the slow dissociation phase. Instead, two more rapid dissociation rates (km1 = 8.53 x 10m3 s-' and 4.43 x lo-' s-') were observed. These results suggest that [3H]bradykinin interacts with at least two B2 kinin receptor-like binding sites in bovine myometrial membranes.
A three-state model for the guanine nucleotide-sensitive agonist interaction with the high affinity binding sites is proposed.
Kinins, a group of endogenous vasoactive peptides with remarkable pharmacological potency, have been implicated in a number of physiological as well as pathophysiological conditions. Kinins contract and relax a number of smooth muscles of both vascular and nonvascular origin (1). At the cellular level, kinins stimulate inositol phospholipid turnover and intracellular Ca2+ mobilization (Z-6), prostaglandin release (7,8), and promote both membrane depolarization and hyperpolarization (9,10). Receptors for kinins have been divided into two main subtypes designated Bl and B2 (1). The B2 receptor subtype is the most prevalent form of the two and appears to mediate most kinin actions under nonpathological conditions. B2 kinin receptors have been identified in uitro on intact cells (11)(12)(13) and in various membrane preparations (14)(15)(16)  A number of agonists bind to their respective receptors in a guanine nucleotide-sensitive manner. This sensitivity is due to the presence of a guanine nucleotide regulatory protein (Gprotein) as an integral component of the receptor-effector complex with high affinity for guanine nucleotides and that serves to transduce the signal elicited by agonist binding from the receptor to the effector component (23). Several reports suggest that both hormone-promoted polyphosphoinositide hydrolysis and release of arachidonic acid metabolites are regulated by specific G-proteins (24,25). The properties of these putative G-proteins are presently unknown. Kinin peptides stimulate both of the above cellular responses. Consequently, we were interested in evaluating the effect of guanine nucleotides on agonist binding to B2 kinin receptors to determine if a G-protein may be involved in B2 kinin receptor signaling. Here

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Guanine Nucleotide Regulation of B2 Kinin Receptors branes and assessed its sensitivity to guanine nucleotides. The experimental procedures and results are described in the Miniprint.
At equilibrium [3H]BK interacted with these membranes with at least two affinities.
Furthermore, the total specific binding displayed a typical B2 kinin receptor specificity. These results indicate that at least two distinct B2 kinin receptor-like binding sites may be present in this tissue. The interaction of [3H]BK with the high affinity binding sites involved a guanine nucleotide-sensitive sequential formation of binding states with decreasing dissociation rates for [3H] BK. The presence of multiple binding states of the high affinity B2 kinin receptor-binding sites may in part be a consequence of coupling to a guanine nucleotide regulatory protein.
The data presented suggests that the low affinity bovine myometrial B2 kinin receptor sites exist in a single binding state (Equation 1) and the high affinity receptor sites exist in at least three binding states (Equation 2). One model for the formation of such states for each receptor site is described below where BK (L) interacts with the low affinity receptor (RL) to yield one complex (LRL) and with the high affinity receptor (Rn, Rfi, or R&*) to yield three complexes (LRn, LR& or LRk). KL represents the equilibrium association constant for the interaction with the low affinity receptor and Kn, -Km represents equilibrium association constants for the interaction with the high affinity receptor. The following discussion is the rationale for proposing the above models.
The high and low affinity binding sites were studied con- The use of guanine nucleotides provided significant additional information regarding the characteristics of agonist binding to the high affinity binding sites. Even though conclusive evidence would require an assay of the low affinity sites without the interference by the high affinity sites, we did not observe any effect of guanine nucleotides on the binding of [3H]BK to the low affinity sites. When the high affinity sites were assayed essentially independently of the low affinity sites, we observed that dissociation of equilibrium binding of [3H]BK proceeded with two phases in the presence of guanine nucleotides rather than with one phase as observed in the absence of guanine nucleotides. The rate of one of these two phases (k-1 = 8.53 x 10e3 s-') is similar to the fast rate observed prior to equilibrium in the absence of guanine nucleotides (k-, = 3.82 x 10m3 s-l). The other phase has a rate (k-1 = 4.43 x 10e4 s-') of a magnitude in between that of the fast (k-1 = 3.82 x 10e3 s-') and slow (k-l = 4.43 X 10e5 s-l) rates. Interestingly, the shallowness of pre-equilibrium dissociation in the absence of guanine nucleotides suggests the presence of other transient pre-equilibrium dissociation phase with values between that of the fast and slow rates and similar to that of the slow dissociation rate observed in the presence of guanine nucleotides. These results support the presence of two pre-equilibrium binding states of the high affinity sites. One state, termed LRn, exhibits a relatively fast dissociation rate, and a second state, termed LRA, exhibits a dissociation rate between that of the fast and slow rates and accumulates only in the presence of guanine nucleotides. Thus, guanine absence of guanine nucleotides, this model is also likely. nucleotides completely prevent the formation of LR$*. On the A two-state model has been used to describe the interaction other hand, LRn and LR$ can still form.
of agonists with a number of receptors (28-33). Our results In the presence of guanine nucleotides [3H]BK binds to suggest that a third binding state (LR&) is involved in BK myometrial membranes with two affinities (37 and 444 PM).
binding to high affinity B2 kinin receptors. We do not yet The relative level of the higher affinity binding observed in know the role of such a putative state. One possible explanathe presence of guanine nucleotides (12%) does not corre-tion is that this represents a state of the receptor which is spond to that of high affinity binding observed in the absence coupled to a G-protein but in a manner that does not promote of guanine nucleotides (45%). The dissociation kinetics indi-an effective functional outcome. Such a state may be required cate that in the presence of guanine nucleotides a significant for the ultimate effective functional coupling of the receptor portion of the high affinity binding sites exist in a rapidly and the G-protein. An analogous state has been proposed for dissociating state (LRn). Thus, based on the above assump-the myocardial muscarinic receptor-G-protein complex (34). tion, the high and low affinity binding observed in the pres-Interestingly, detergent extraction of guanine nucleotide-senence of guanine nucleotides probably represent LRf: and LRn sitive [3H]BK binding does not require prior exposure of + LRt, respectively. Following this assumption, 12% of the membranes to bradykinin.3 Hence, a "pre-coupled" form of total binding exists as LR&. As indicated by the dissociation the receptor may either exist or may easily form in the kinetics, the residual portion of the high affinity binding must membrane under some conditions. represent a binding state with rapid dissociation kinetics.
Based on dissociation rate constants as well as apparent This is supported by the presence of a significant amount of equilibrium dissociation constants for [3H]BK, HRn and HRL rapidly dissociating binding in the presence of guanine nucle-may have similar binding characteristics. Furthermore, both otides. Since we cannot clearly distinguish LRn and LRL, we high and low affinity binding sites display typical B2 kinin cannot directly determine the relative level of LRn.
receptor specificities. We have no information about the At 0.05 nM, Gpp(NH)p maximally inhibited binding ap-biochemical differences between the high and low affinity proximately 50%. Using the experimental values of the rela-sites. However, we know from sucrose gradient sedimentation tive levels of the high and low affinity binding sites and the studies of soluble binding activity that the sedimentation rate KD values for each of the receptor states, at 0.05 nM [3H]BK of at least a portion of the molecular species that [3H]BK 66% of the binding should be sensitive to guanine nucleotide. binds to in bovine myometrial membranes decreases following This value is close to the experimental value above and shows exposure to G~~(NH)P.~ It is conceivable that the high and that the kinetic parameters determined for each binding site low affinity binding site represent forms of B2 receptors that are essentially correct. are able and unable, respectively, to couple to a G-protein.
In summary, the apparent monophasic dissociation kinetics The two binding sites may represent distinct subtypes of the of BK binding to RL suggest that Rt forms a single binding B2 receptor, and their presence in our preparation may be a state (Equation 1). The lack of dependence of the association consequence either of their coexistence in bovine myometrial rate on low concentrations of BK indicates that the high tissue or from the fact that one of them exists in a tissue, e.g.
affinity binding reaction is complex and involves multiple endometrium, that is contaminating our preparation. The kinetic states. One possibility is that the interaction of BK presence of B2 bradykinin receptor subtypes has been proand the receptor initially yields a binding complex(es) from posed (16,(18)(19)(20)(21)(22).
which BK dissociates rapidly and which we have termed LRn Odya et al. (35) reported that the angiotensin converting  (36) formation of LR$* is completely dependent on prior formation and has been proposed to be a consequence of a coupling of of LR&. However, LR&* is clearly in equilibrium with the these receptors with a G-protein. Agonist-promoted inositol medium as it exhibits a dissociation rate distinct from Rn and phospholipid metabolism and arachidonic acid release has R&. These results suggest that LRi$* probably represents the been proposed to involve a G-protein. Cholera toxin and state of the high affinity bovine myometrial B2 kinin receptor pertussis toxin, which ADP-ribosylate specific G-proteins, that is effectively coupled to a G-protein.
have served as tools to probe the involvement of G-proteins Another model is where LR$ represents an alternative state in these processes. BK-promoted inositol phosphate producthat LRn partially proceeds to and equilibrates with in the tion and arachidonic acid release in A431 epidermoid carciabsence of LR$* formation (in the presence of guanine nucle-noma cells (4), bovine pulmonary artery endothelial cells (6), otides) rather than an intermediate state in the sequence of and Swiss 3T3 fibroblasts (8) were not sensitive to pertussis events leading to formation of LRg*. In the absence of evi-toxin while bradykinin-promoted hyperpolarization and dedence for triphasic pre-equilibrium dissociation curves in the polarization in NG108-15 neuroblastoma-glioma hybrid cells were inhibited (2). Thus, the B2 kinin receptor may be linked to different G-proteins depending on the cell type which it is located in and/or which second messenger system it is associated with. We have shown that bovine myometrial membranes contain high and low affinity binding sites for [3H]BK which display a pharmacological specificity typical of a B2 kinin receptor. The high affinity receptors are sensitive to guanine nucleotides suggesting that they can couple to a guanine nucleotide regulatory protein. We propose that formation of a G-proteinreceptor coupling involves the sequential formation of at least three receptor-binding states with decreasing dissociation rates for [3H]BK.