Perspectives in Pharmacology Stoichiometry and Compartmentation in G Protein-Coupled Receptor Signaling: Implications for Therapeutic Interventions

There is great therapeutic interest in manipulating (either enhancing or suppressing) G protein-coupled receptor (GPCR) signal transduction. However, most current strategies are limited to pharmacological activation or blockade of receptors. Human gene therapy, including both overexpression and antisense approaches, may allow manipulation of GPCR signaling at steps distal to receptors. To fully understand the impact of such therapy, the transduction of signals between the multiple components of GPCR signaling and their interaction with other cellular molecules must be understood in the context of both normal physiology and disease. Defining the stoichiometric relationship among multiple components of GPCR signaling is a first step. We summarize data showing the substantial excess of Gas relative to both b-adrenergic receptors and adenylyl cyclase. A predominant idea regarding signaling via GPCRs has for over 20 years emphasized the concept of random movement and collision (“collision coupling”) of proteins within the lipid bilayer of the plasma membrane. This notion does not readily account for the rapidity and fidelity of signal transduction by the multiple components involved in GPCR-G proteineffector systems, especially considering the low abundance of these proteins in cells. Recently, many components involved in signal transduction by GPCRs have been shown to exist primarily in microdomains of the plasma membrane, in particular, caveolae. These and other structures may serve to compartmentalize signals, thereby optimizing signal transduction between an agonist and specific effectors. The formation, organization, and maintenance of such structures may prove to be altered in disease states associated with disregulated signaling. In addition, we speculate that identification of genetic polymorphisms of and therapy targeted to components that are critical for determining efficacy (e.g., effectors such as adenylyl cyclase) will provide important future therapeutic strategies. The transduction of signals from the extracellular environment across the plasma membrane barrier and into the intracellular milieu is a fundamental aspect of cellular regulation. Nature has evolved a variety of means to accomplish this feat, in particular, via the use of many different types of ligands and receptors. One can generalize that such signal transduction pathways fall into four basic paradigms: 1) membrane receptors that function as ion channels, 2) membrane receptors that are enzymes, 3) intracellular receptors that recognize lipophilic ligands that diffuse across the plasma membrane, and 4) G protein-coupled receptors (GPCRs). In contrast with the other three systems, GPCR systems involve membrane interaction of components in addition to the receptor to initiate transduction of extracellular signals into the cell. Additional molecules are required to mediate feedback regulation and to integrate such signals with other cellular inputs and events. Therapeutic manipulations of GPCR systems have thus far been limited primarily to pharmacological blockade or activation of the receptors. Although GPCRs are useful as drug targets because of their patterns of distribution on different cell types and the prefReceived for publication February 10, 2000. 1 This work was supported by grants from the National Institutes of Health and the Cystic Fibrosis Foundation. ABBREVIATIONS: GPCR, G protein-coupled receptor; AC, adenylyl cyclase; RGS, regulator of G protein signaling; AC6, adenylyl cyclase type 6; PGE2, prostaglandin E2; PKA, cAMP-dependent protein kinase; AKAP, A-kinase anchoring protein; PKC, protein kinase C; AR, adrenergic receptor; GRK, G protein receptor kinase; RACK, receptor for activated C kinase; RAMP, receptor activity modifying protein; PLC, phospholipase C. 0022-3565/00/2942-0407$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 294, No. 2 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 294:407–412, 2000 /900008/834822 407 at A PE T Jornals on July 2, 2017 jpet.asjournals.org D ow nladed from erential role of particular GPCR subtypes in mediating specific responses, postreceptor components are also potential therapeutic targets. If one wishes to alter GPCR signaling pathways in novel ways, it is necessary to understand the dynamics of activation for each component in the pathway and the subsequent interactions among these components. One approach to identify novel therapeutic strategies is to examine the stoichiometry, i.e., absolute concentrations or relative proportions of each component, of a GPCR signal transduction pathway expressed in a given cell. Identifying the components that determine potency (sensitivity, EC50, etc.) and efficacy (maximal response) can lead to insights as how to best enhance or suppress a disregulated system. Such studies have been completed for the Gs-linked adenylyl cyclase (AC) pathway in cardiac myocytes (described below). Moreover, the recent evidence that many signaling molecules are enriched in specialized microdomains of the plasma membrane increases the likelihood that GPCR signaling is highly compartmentalized in cells (for reviews, see Neubig, 1994; Chidiac, 1998; Okamoto et al., 1998; Shaul and Anderson, 1998). Considering the rapidity and fidelity of signal transduction by GPCR systems, it has been suggested that the essential molecules of such pathways are held in close association with one another and not freely floating or dependent on random collision to interact. The evidence supporting this idea and the therapeutic implications of stoichiometric expression and compartmentation are the focus of this Perspective. Components of GPCR Signaling: GPCR-Gs/GiAC as a Paradigm In addition to GPCRs as the initial components that interact with extracellular hormone or neurotransmitter, GPCRs transduce signals by coupling to heterotrimeric (a-, b-, g-subunit-containing) GTP-binding (G) proteins that regulate effector molecules. There are four principal G protein families (Gs, Gi, Gq/11, G12/13), each identified by structurally similar a-subunits that preferentially regulate specific classes of effector molecules. Gq/11 stimulates phospholipase C (PLC), Gs stimulates AC, and Gi inhibits AC and activates K 1 channels, although family members can regulate multiple types of effector enzymes, ion channels, and transporters. The intrinsic GTPase activity of Gi and Gq a-subunits can be enhanced by regulators of G protein signaling (RGS) proteins (Dohlman and Thorner, 1997), and Gs a-subunit GTPase activity can be enhanced by AC (Scholich et al., 1999). The Gb and Gg subunits function as a heterodimer to regulate effector molecules and other proteins involved in GPCR signaling and also to restrain Ga action by forming inactive Gabg complexes. Among the various G protein-regulated effectors, AC is arguably the most well studied and has provided a particularly useful system to examine GPCR stoichiometry. AC, regulated by Gs and Gi, synthesizes cAMP from ATP, which in turn regulates cell function via activation of cAMP-dependent protein kinase (PKA). PKA phosphorylates serine residues on substrates to initiate cellular actions of cAMP, and phosphatases reverse such phosphorylation and actions. Cells “target” the relatively nonspecific kinase activity of PKA via A-kinase anchoring proteins (AKAPs) so that the kinase preferentially phosphorylates specific substrates (Colledge and Scott, 1999). A substantial number of different AKAP proteins have been identified. These include molecules that show preference for individual isoforms of PKA and for interaction with specific types of substrates. An example is AKAP250 (also known as gravin), which interacts with the b-adrenergic receptor (b-AR) and targets activated PKA to phosphorylate the receptor, thus permitting specific feedback regulation of receptor activity (Shih et al., 1999). Targeting mechanisms also exist for G protein receptor kinases (GRKs) that phosphorylate b-ARs and other GPCRs. Targeting of GRK to activated receptors is mediated by Gbg subunits, which appear to enhance the specificity of GRK for agonistoccupied receptors (Krupnick and Benovic, 1998; Lefkowitz, 1998). By phosphorylating receptors, GRKs impair interaction of GPCRs with G proteins and induce the recruitment of b-arrestin, a protein that acts as an adapter between the receptor and clathrin and thereby initiates internalization via clathrin-coated pits (Ferguson et al., 1996; Goodman et al., 1996). Stoichiometry of AC Pathway As discussed above, there is great interest in therapeutic efforts to modulate GPCR signaling. This has been particularly true for b-AR signal transduction. Given the multicomponent interactions required for GPCR signaling, it is intriguing to imagine that each of the components has the potential to be a therapeutic target. Developing new approaches to accomplish this depends on understanding which component(s) of these signal transduction pathways are most critical in determining efficacy and potency of the system. Classical receptor theory predicts that expression of receptor, as the site of interaction with the agonist, determines potency, and indeed some studies confirm this theoretical prediction (Milligan, 1996). However, it is not so simple to prognosticate which component determines efficacy of the system. In part, this difficulty relates to what one may wish to define as “response”. Most molecular pharmacologists focus on the initial event (i.e., generation of second messenger) and assess receptor occupancy relative to maximal formation of second messenger. We emphasize this approach in this Perspective. Others assess response as activation of “downstream” enzymes, channels, or physiological events. In these latter cases, downstream components that limit the rate or extent of response may prove to be as, or more, critical than upstream components that regulate second-messenger formation. For example, if one wishes to examine b-AR activation in the heart, one might relate receptor occupancy to G protein or AC activation, cAMP formation, PKA activation, ion channel activity, contractile protein or metabolic enzyme phosphorylation, or measures of inotropy, chronotropy, lusitropy, or dromotropy. Studies to assess efficacy based on second-messenger formation necessitate quantification of each of the components involved. In the case of b-AR-Gs-AC, one can use radioligand binding to quantitate receptors and AC (using [H]forskolin for the latter) and radioimmunoassay or quantitative immunoblotting to quantitate Gs. Using this approach, we conducted initial studies to quantify b-AR signaling components of AC in S49 murine lymphoma cells. In these cells, the ratio of receptor:G protein:AC is approximately 1:100:3, and receptor activation of Gs appears to be the critical factor for am408 Ostrom et al. Vol. 294 at A PE T Jornals on July 2, 2017 jpet.asjournals.org D ow nladed from plification of signaling (Alousi et al., 1991). Subsequent studies in adult rat cardiac myocytes and with NG108-15 cells yielded comparable results (Post et al., 1995; Milligan, 1996). These results, together with assumptions regarding uniform accessibility and equivalent function of individual components, lead one to predict that either receptor or AC, but not Gs, would set the limit on the maximum efficacy of the system. Related data are consistent with the idea that other effectors (e.g., voltage-sensitive calcium channels) are expressed at a level akin to that of AC (Szabadkai et al., 1998). How the low absolute concentration of GPCR, G protein, and effector (in the femtomole to low picomole per milligram of protein range) favors rapid, high-fidelity interaction of components required for signal transduction in native cells and membranes is not clear. In addition, the stoichiometric expression of bg-subunits, in particular different combinations of band g-subunits, has not been evaluated carefully relative to other components, a major shortcoming considering their importance in GPCR signaling. Release of bg-subunits should occur in molar equivalent to that of a-subunits on activation, but their subsequent cellular effects likely depend on the regulation of effectors by specific isoforms of bg (Hilde-