G-Protein Coupled Receptor Resensitization – Appreciating the Balancing Act of Receptor Function

Abstract

G-protein coupled receptors (GPCRs) are seven transmembrane receptors that are pivotal regulators of cellular responses including vision, cardiac contractility, olfaction, and platelet activation. GPCRs have been a major target for drug discovery due to their role in regulating a broad range of physiological and pathological responses. GPCRs mediate these responses through a cyclical process of receptor activation (initiation of downstream signals), desensitization (inactivation that results in diminution of downstream signals), and resensitization (receptor reactivation for next wave of activation). Although these steps may be of equal importance in regulating receptor function, significant advances have been made in understanding activation and desensitization with limited effort towards resensitization. Inadequate importance has been given to resensitization due to the understanding that resensitization is a homeostasis maintaining process and is not acutely regulated. Evidence indicates that resensitization is a critical step in regulating GPCR function and may contribute towards receptor signaling and cellular responses. In light of these observations, it is imperative to discuss resensitization as a dynamic and mechanistic regulator of GPCR function. In this review we discuss components regulating GPCR function like activation, desensitization, and internalization with special emphasis on resensitization. Although we have used β-adrenergic receptor as a proto-type GPCR to discuss mechanisms regulating receptor function, other GPCRs are also described to put forth a view point on the universality of such mechanisms.

INTRODUCTION

Cells respond to a wide range of environmental cues through a plethora of signals that are physical, chemical, and/or biological in nature. The plasma membrane acts as a barrier and sensor for the majority of these extracellular stimuli providing an appropriate cellular response using cell surface receptors. Among various types of cell surface receptors, G-protein coupled receptors (GPCRs) constitute a major and most ubiquitously expressed class of receptors. GPCRs are named so by virtue of their interaction with guanine nucleotide binding regulatory proteins (G-proteins) following receptor activation. GPCRs are the largest super family of seven transmembrane receptors representing nearly 4% of all the proteins encoded in the human genome [1]. GPCRs regulate a multitude of biological processes like neurotransmission, cardiac function, vision, olfaction; they are thus the most targetable surface molecules accounting for about 30-40% of the currently prescribed drugs [2, 3]. Therefore, it is implausible to study and analyze nearly any cellular function without considering the role of GPCRs in biology.

The transduction of the extracellular stimuli from outside of the cell to inside is mediated by cyclical regulation of GPCRs. These cyclical events involve activation, desensitization, and resensitization of GPCRs. Following activation of the GPCRs by an agonist, the receptors are phosphorylated resulting in desensitization. The desensitized receptors are targeted for internalization into the endocytic compartments. The desensitized endocytosed receptors are recycled back to the plasma membrane following resensitization by dephosphorylation in the endocytic vesicles (Fig. 1) [4-6]. In addition to this classical paradigm which reckons that internalization is a prerequisite step in resensitization, increasing number of studies have suggested that resensitization of GPCRs could occur at the plasma membrane without internalization [7-11]. It is also known that a subset of internalized receptors traffic to the late endosomes for degradation. Among the steps of receptor regulation, mechanisms of activation and/or desensitization have been discussed extensively [12-14] with little emphasis on resensitization. Recent studies suggest that resensitization is not a steady state normalizing event but an acutely regulated process, alterations of which could affect receptor signaling. In appreciation of this changing paradigm, it is critical to discuss the shifting view due to the progress in understanding resensitization. In the review we will provide an overview on the regulation of GPCR function with a unique stress on receptor resensitization. We have used the β-adrenergic receptors as a proto-typical model system and have included studies from other GPCRs in the review to put forward the idea that these mechanisms could be universal.

Fig. (1). Classical view of desensitization, resensitization, and recycling of GPCRs.

When a ligand binds to the GPCR it causes a conformational change in the GPCR. The GPCR activates an associated G-protein by exchanging its bound GDP for a GTP. The G-protein's α subunit dissociates from the β and γ subunits to further affect intracellular signaling proteins or target functional proteins. The dissociated βγ subunits facilitate the recruitment of GRK and PI3K to the receptor complex. The GRK phosphorylates C-terminal tail of the receptor which results in increased affinity and binding of the receptors to β-arrestins. Once bound, β-arrestins prevent G-protein coupling (desensitization) and may recruit other proteins AP-2 and clathrin required for receptor endocytosis (internalization). PI3K plays a role in generation of D3 phosphoinositides required for recruitment of AP-2 to the receptor complex. The membrane buds inwardly and pinched off from the membrane to form endocytic vesicles. Once internalized the receptors are trafficked to recycling endosomes or targeted to lysosomes for degradation. The receptors in the recycling endosomes are subsequently dephosphorylated by PP2A and recycled back to the plasma membrane as resensitized receptors.

GPCR SIGNALING

Ligand binding to GPCRs results in conformational change that activates its cognate hetero-trimeric G-protein composed of G_α and G_βγ subunits. Activation of hetero-trimeric G-protein initiates the exchange of bound GDP for GTP by G_α subunit leading to dissociation of the heterotrimer into G_α and G_βγ subunits. Dissociation of the G_α and G_βγ subunits results in generation of second messengers leading to downstream signaling. This represents the classical paradigm of G-protein dependent signaling, in which it was initially believed that only the dissociated G_α subunit is the functional component with G_βγ being a passive player in signaling [15]. Subsequently, studies identified that G_βγ subunits of the G-proteins play as important a role in regulating downstream signals as G_α subunit [16]. G_βγ subunits play a critical role in recruiting G-protein coupled receptor kinase (GRK) to the GPCR complex [6, 17] that mediates receptor phosphorylation [18, 19] resulting in recruitment of scaffolding protein β-arrestin [20, 21]. β-arrestin physically interdicts further G-protein coupling [22] and targets the receptor towards internalization [23, 24]. In addition to the classical G-protein dependent signaling, the recruited β-arrestin can initiate G-protein independent signaling from the receptor complex [25-27]. This changing landscape of receptor signaling has been very well discussed in recent reviews [27-30], but the regulation of GPCR resensitization has not been accorded its due importance. Therefore, in the current review we have provided a perspective on GPCR resensitization and its effects on receptor signaling potentially contributing towards physiology and/or pathology.

GPCR DESENSITIZATION

Classically, desensitization of the GPCRs is described as phosphorylation and functional uncoupling of the receptor from the cognate G-protein [31]. This striking inability of the receptors to couple to G-proteins depicting desensitization is observed in various pathologies including heart failure and asthma [31-34] wherein β-adrenergic receptors are desensitized [32, 33]. GPCR desensitization is initiated via the phosphorylation of activated receptors by the family of seven GRKs [30]. Among the seven GRKs, GRK 2, 3, 5, and 6 are ubiquitously expressed while GRK 4 is localized in the testis, and GRK 1 and 7 in visual systems [4, 18, 35, 36]. GRK 2, 3, 5, and 6 play a major role in regulating GPCR responses to stimuli and stress due to their ubiquitous distribution. GRK 2 and 3 are cytosolic in distribution and translocate to the receptor complex following agonist activation [5]. The translocation and membrane localization of GRK 2/3 are mediated by their binding to G_βγ subunits via the pleckstrin homology domain [37]. In contrast, GRK 4, 5, and 6 are constitutively localized to the plasma membrane. GRK 4 and 6 are palmitoylated allowing for membrane association [38-40] while GRK 5 contains polybasic domains that mediate electrostatic interactions with membrane phospholipids [41-43]. Despite their varied mechanisms of interaction with the membrane, underlying function of these GRKs is to phosphorylate agonist activated receptors initiating desensitization.

Extensive in vitro studies have shown that GRK 2 can phosphorylate most GPCRs [36] and is the major isoform expressed in many cells. The importance of GRK 2 in regulating GPCR function can be appreciated by the finding that genetic deletion of GRK 2 is embryonic lethal [44]. However, cardiac specific overexpression of GRK 2 in mice attenuates β-adrenergic receptor and angiotensin II type 1 receptor signaling with no desensitizing effect on adenosine or α1-adrenergic receptors [45, 46]. Although GRK 2 and 3 are highly homologous and regulated by G_βγ subunits, overexpression of GRK 3 does not alter cardiac β-adrenergic receptor function [45]. Interestingly, GRK 3 is involved in desensitization of α1B-adrenergic and thrombin receptors [45]. Consistent with differential regulation of receptors by GRKs, GRK 5 desensitizes β-adrenergic and adenosine receptors but not angiotensin II type 1 receptor [46]. These observations support the idea that GRKs may regulate different receptors in a temporal manner indicating that more needs to be understood about GRK-GPCR specificity. In addition to receptor specificity, there is yet another layer of receptor regulation that involves differential phosphorylation of a single GPCR by different GRKs. Particularly, serine or threonine residue phosphorylated by individual GRK on a GPCR may determine which specific downstream signaling pathway is activated. For instance, phosphorylation of angiotensin II type 1 receptor by GRK 5 or 6 activates downstream extracellular signal regulated kinase (ERK) and phosphorylation by GRK 2 or 3 results in endocytosis [47]. Based on the observation that each of the GRKs could specifically phosphorylate distinct sites on the GPCR, the idea of “barcode” or a specific GPCR phosphorylation pattern has been proposed [48]. This specific “barcode” may dictate the structural confirmation assumed by bound β-arrestins that may in turn provide unique β-arrestin scaffolding surfaces for interaction with downstream signaling complexes influencing functional manifestation [47, 49].

Recruitment of β-arrestin to the receptor complex is considered critical and the final element in GPCR desensitization. β-arrestin binding to the GPCRs physically interdicts further G-protein coupling [22] completing the receptor desensitization. β-arrestin blocks the access of G-proteins to the GPCR binding domains thereby reducing the ability of hetero-trimeric G-protein to dissociate into G_α and G_βγ subunits decreasing downstream signals initiated by the G-proteins [21]. Furthermore, β-arrestins act as scaffolding proteins for enzymes like phosphodiesterases and diacylglycerol kinases providing an enriched platform for signaling [50, 51].

GPCR INTERNALIZATION

Majority of the cell surface receptors including GPCRs undergo endocytosis upon agonist stimulation resulting in removal of the receptors from the plasma membrane [5, 24, 52]. In addition to initiating desensitization of GPCRs, β-arrestin targets the receptor towards endocytosis by recruiting components mediating GPCR internalization [53]. This role of β-arrestin is clearly dependent on GRK mediated phosphorylation of the receptors. Indeed, GRK phosphorylation site deficient β-adrenergic receptors have poor ability to recruit β-arrestins to the receptor complex resulting in marked reduction of receptor internalization [54]. The role of β-arrestin in GPCR internalization has been unequivocally demonstrated using dominant negative strategy [23], mouse embryonic fibroblasts from β-arrestin 1/2 double knock out mouse embryos [55] and siRNA knock downs [56]. β-arrestin mechanistically coordinates endocytosis of GPCRs by recruiting critical components of the endocytic machinery [21, 30, 53]. β-arrestin predominantly recruits components of the clathrin mediated pathway to the GPCRs targeting the receptor for internalization using the clathrin coated vesicles [21, 30, 53].

In the clathrin coated vesicle mediated internalization, β-arrestin recruits AP-2 adaptor and clathrin molecules to the receptor complex that form the invaginated basket at the plasma membrane. Thus, β-arrestin acts as a bridge between the GPCRs and the building blocks of the clathrin coated pit AP-2. The AP-2 complex, which is a hetero-tetramer of adaptin proteins, tethers clathrin triskelion to form the woven basket [57-59] that is targeted for internalization in a regulated manner. The clathrin coated pits formed on the plasma membrane in coordination with dynamin pinch off as clathrin coated vesicles culminating in internalization of the cell surface GPCRs [60]. Dynamin is a small GTPase that is critically responsible for endocytosis in eukaryotic cells and mutation in this protein causes aberrant GPCR endocytosis [61]. Although β-arrestin mediated endocytosis of GPCRs via clathrin coated vesicles is a major pathway of internalization, there exist β-arrestin independent alternate mechanisms of internalization like endocytosis through caveolae, uncoated pits, or lipid rafts [53].

In addition to these critical molecules that are the nuts and bolts (components) of endocytosis, there are many other molecules that modulate the process of internalization by regulating the components of the endocytic machinery. One such molecule is phosphoinositide 3-kinase (PI3K)γ that plays a critical role in recruitment of AP-2 to the β-adrenergic receptor complex [62]. PI3Kγ is recruited to the receptor complex following agonist activation and generates D3 phosphoinositides which mediate effective localization of AP-2 adaptor protein to β-adrenergic receptors driving internalization [62]. Protein kinase activity of PI3Kγphosphorylates non-muscle tropomyosin mediating localized actin remodeling critical for movement of clathrin coated vesicles [63]. Another important molecule in vesicular trafficking of GPCRs is the ADP-Ribosylation Factor 6 which is a member of the ADP-Ribosylation Factor family of small GTP binding proteins [64]. Activation of the ADP-Ribosylation Factor 6 occurs by exchange of the GTP for GDP which is catalyzed by guanine nucleotide exchange factor ARNO (ADP-Ribosylation Factor Nucleotide binding site Opener). ARNO is constitutively associated with β-arrestins [65] acutely regulating vesicular traffic. ADP-Ribosylation Factor is also regulated by the GTPase activating protein that accelerates GTP hydrolysis resulting in inactivation of the ADP-Ribosylation Factor 6. The GRK interacting protein (GIT) is thought to be the GTPase activating protein for ADP-Ribosylation Factor 6 as overexpression of GIT results in reduced β-adrenergic receptor internalization [66-68]. Interestingly, the activity of GIT proteins is regulated by phosphatidylinositol 3, 4, 5-triphosphate [67] raising exciting possibilities of a regulatory link between ADP-Ribosylation Factor 6 and PI3Kγ in the process of receptor internalization.

Although the scaffolding function of β-arrestin orchestrates receptor internalization, there are also other proteins such as PSD95 and MAGI-2 that regulate receptor internalization. Overexpression of PSD95 decreases β-adrenergic receptor internalization while overexpression of MAGI-2 results in enhanced agonist-induced internalization [69, 70]. Currently, the underlying mechanism of this antagonizing action is not yet fully understood. An important caveat in all the above mentioned studies is that they provide the intricate mechanism of activation, but little is understood in terms of processes regulating the inactivation of the activated proteins. We believe that such mechanisms exist in vivo as activation or inactivation is attained biologically by a shift in dynamic equilibrium, thereby increasing the representation of the molecules in any one of these states. Such a view suggests that regulation of GPCRs though well understood, is yet incomplete and it would not be surprising if new regulators emerge or novel functions are added to the existing repertoire of molecules. Slowly but surely efforts have been invested to bridge these knowledge gaps. For instance, all the efforts have been concentrated on how β-arrestin scaffolds proteins involved in receptor endocytosis but it is not clear how this complex dissociates after execution of its functions. Recent studies show that Src (a tyrosine kinase) mediated phosphorylation of AP-2 regulates dissociation of AP-2/β-arrestin complex [71, 72]. We believe that with time and effort better understanding of the process will be achieved and the novel observations will be integrated into the paradigm of receptor regulation.

GPCR RESENSITIZATION

Once the activated GPCR is desensitized by phosphorylation and internalized, the receptor can follow different intracellular trafficking routes. Desensitized GPCRs can be sorted to the lysosomes for degradation or dephosphorylated in the endocytic compartment for recycling to the cell surface for agonist stimulation [5]. Sorting of the GPCRs to recycling or degradation pathways is a critical step before receptor resensitization and thus has been of intense interest. Studies have identified that multiple events regulate the process of trafficking/sorting. This machinery can be predominantly categorized into proteins that bind to the receptor complex to determine GPCR fate or post-translational modifications of the receptor that channel the complex to a specific pathway [5, 73-75]. The plethora of studies point to the complex nature of this process and our understanding of its regulation is incomplete.

Recycling and GPCR Resensitization

In addition to playing a pivotal role in GPCR desensitization and internalization, β-arrestin also regulates trafficking of receptors which is a critical step prior to resensitization. β-arrestin binds to the GPCRs with different affinities [76, 77] and receptors can be divided into two classes (A and B) based on relative stability of the β-arrestin-receptor complex. Class A GPCRs preferentially bind to β-arrestins with low affinity, and as a result fall off from the receptor prior to undergoing internalization into the endosomes. In contrast, Class B GPCRs have higher affinity for β-arrestins forming a stable β-arrestin-receptor complex resulting in internalization of the complex into the endosomes. This differential binding of β-arrestin to the GPCRs and its targeting for internalization is regulated by a serine cluster at the C-terminal region on the receptors. Thus by changing the C-terminal serine cluster, a Class A receptor can be converted to Class B receptor [78]. The GPCRs that belong to Class A are efficiently dephosphorylated, resensitized, and rapidly recycled back to the plasma membrane. While Class B receptors that bind β-arrestin with high affinity and internalize with bound β-arrestin are less efficiently recycled delaying the trafficking of the receptors back to the plasma membrane [4, 12].

Apart from β-arrestins, there are also other proteins that play a critical role in trafficking of the GPCRs. The Rab GTPases are recognized as regulators of vesicular membrane transport/sorting. Differential function of Rab 4 and Rab 5 in trafficking of receptors is considered to be an integral step in receptor resensitization. Dominant negative Rab 4 expression in cells blocks β-adrenergic receptor recycling [79] while Rab 4 inhibition in vivo prevents resensitization after isoproterenol-induced β₂-adrenergic receptor desensitization [80]. Dominant negative and constitutively active Rab 5 strategies shows that it is a critical player in β₂-adrenergic receptor internalization but not in resensitization [79]. More studies are required to determine the underlying mechanism(s) of Rab GTPase mediated receptor resensitization.

Internalized receptors could either be recycled back to the plasma membrane or trafficked to lysosomes for degradation, and mechanisms regulating the targeting of receptors to these distinct pathways are not well understood. The binding of Na⁺/H⁺-Exchanger Regulatory Factor to β₂-adrenergic receptors results in efficient recycling as disruption of this interaction results in agonist dependent lysosomal degradation [75]. Thus, we believe that with increasing interest in recycling and resensitization, new molecules or new roles for well known molecules will be discovered that may be as important or if not, more critical than those currently identified. An important caveat that needs to be emphasized here is that all the processes that are mentioned till now including receptor desensitization, internalization, and recycling could all be reversible at various stages. As these stages are regulated, it is plausible that studies may identify molecules that may play a role in reversing these steps in a regulated manner but are inhibited following agonist activation of the receptor. We believe that agonist activation sets a stage for the shift in the dynamic equilibrium towards desensitization and internalization.

The functionally complex yet intricate process of resensitization not only involves regulation by proteins binding to the receptor complex but also the state of post-translational modification on the GPCRs. One of the most well studied modifications on the GPCRs is ubiquitination as it governs the pathway of internalized receptors. The ubiquitination of GPCRs determines whether endocytosed receptors take the route of recycling endosomes or lysosomes for degradation [81, 82] as demonstrated for β₂-adrenergic receptors as well as chemokines receptors (CXCR4) [83-86]. Ubiquitination of the receptors is brought about by β-arrestin-mediated recruitment of ubiquitinating enzyme E3 ligase that determines the pathway utilized by the receptors [87]. It is known that reduced β₁-adrenergic receptor ubiquitination is associated with lowered rates of receptor degradation providing a direct relationship between ubiquitination and receptor downregulation pathways [88]. In addition to delivering ubiquitinating machinery to the GPCRs, β-arrestin also recruits deubiquitinases USP20 and USP33 to the receptor [89]. This provides a dynamic environment for the GPCRs to undergo context dependent ubiquitination/deubiquitination that dictates the targeting of the receptors for recycling or lysosomal degradation [5, 21] which is a critical determinant of resensitization.

Ubiquitination may be considered to be a key player in determining the fate of the receptor, but in certain cases de novo synthesis of receptors could play a role in maintaining sensitized state. Such a phenomenon is observed with protease-activated receptors 1 and 2 which are activated by thrombin. These receptors are irreversibly activated by cleavage and internalized for degradation. Therefore, cells have a large pool of intracellular receptors that reside in the Golgi and translocate to the plasma membrane upon cell-surface activation leading to plasma membrane replenishment [90]. Replenishment of GPCRs at the plasma membrane occurs also for δ and ν opioid receptors [91, 92] indicating that this could also be an important contributing player in maintaining sensitivity of cells to specific agonists.

Though irreversible binding of agonist that targets the receptors towards degradation happens with certain GPCRs, majority of the GPCRs are believed to undergo the cyclical process of phosphorylation, dephosphorylation, and recycling to maintain sensitivity in the cells. In this regard, phosphorylation of the GPCRs on specific sites by kinases is known to dictate recycling and resensitization. For example, phosphorylation of β₁-adrenergic receptor on serine 312 on the intracellular loop by protein kinase A is required for efficient resensitization and recycling [93]. This protein kinase A mediated β₁-adrenergic receptor phosphorylation, resensitization, and recycling is in part, regulated by A-kinase anchoring protein 79 (AKAP79). AKAP79 provides platform for protein kinase A to access and phosphorylate the receptor [94, 95]. Similarly, AKAP250 (Gravin) regulates resensitization and recycling of internalized β₂-adrenergic receptors [96, 97]. The mechanism by which Gravin modulates resensitization is through a non-receptor tyrosine kinase Src which provides docking accessibility of Gravin to the receptor [73].

However, an important evolving area of GPCR biology that is yet to be integrated into understanding resensitization is GPCR oligomerization. Expression of β₂-adrenergic receptors along with δ and κ opioid receptors leads to hetero-oligomerization which does not change ligand affinity but alters endocytosis of the oligomers [98, 99]. Furthermore, oligomerization is required for transport of the GPCRs to the plasma membranes altering sensitivity to given set of ligands [100]. Based on the oligomerization, we can postulate that the combination of GPCRs in the complex could determine the recruitment and assembly of scaffold proteins that may regulate GPCR desensitization/resensitization. Particularly, it is important to highlight that the various processes like internalization, endosomal sorting, trafficking, lysosomal degradation, and recycling that have been discussed above are all critical for modulating resensitization. This is based on the paradigm that these steps are interwoven in the scheme of resensitization and altering them thus may have dynamic effects. It is important to note that these steps we have discussed are events that occur prior to or along with the main event of resensitization i.e., receptor dephosphorylation. Alterations in receptor dephosphorylation completely blocks receptor coupling and signaling independent of all the above described events suggesting that GPCR dephosphorylation is a central event piece in resensitization [8, 9, 101].

Dephosphorylation and GPCR Resensitization

Appreciation of GPCR dephosphorylation as a key component in resensitization, though well accepted, is not reflected in the number of published reports. Studies by Krueger et al., show that acidification of the endosomal vesicles is the key determinant in dephosphorylation and resensitization of β₂-adrenergic receptors which is mediated by protein phosphatase 2A (PP2A) [101]. Despite these seminal findings, the process did not garner enough traction due to the prevailing idea that dephosphorylation of the receptors is a passive homeostasis maintaining process and not acutely regulated like kinase-mediated phosphorylation in response to stimulation [15, 22, 102, 103]. Evidence indicates that PP2A function is regulated [104] laying foundation to the idea that PP2A-mediated dephosphorylation may not be a passive homeostasis maintaining phenomenon. Structure of PP2A [105-107] provides a better understanding of PP2A function in various contexts [108, 109] including its role in dephosphorylation of GPCRs.

Increasing interest in understanding GPCR dephosphorylation has led to deeper appreciation that resensitization is not any more synonymous to endosomal sorting and recycling, but a clearly defined independent process. As efforts in the last few decades have been directed towards understanding receptor phosphorylation, internalization, intracellular trafficking, and recycling, little has been determined in terms of the location of GPCR dephosphorylation events. In this regard, the location of GPCR dephosphorylation needs to be discussed as recent studies have shown that β₂-adrenergic receptors in certain conditions can undergo dephosphorylation independent of receptor internalization [110]. β₂-adrenergic receptor undergoes dephosphorylation with low concentrations of isoproterenol which does not induce internalization [110] showing that more studies are required to understand these mechanisms. Such an observation is in contrast to the paradigm on receptor resensitization wherein it is thought that receptor internalization is a prerequisite step in this process. Although these studies suggest a shifting paradigm with regards to the process of resensitization, we believe that it is more complex and context dependent. Such a view point is supported by studies showing differential receptor responsiveness with inhibition of internalization and discussion below provides an appreciation of such context dependent regulation.

Inhibition of internalization completely blocks recovery of A₂ adenosine receptor responsiveness and has no effect on resensitization of inositol phosphate-prostanoid receptor [111]. Adding another layer of complexity to this process is the phosphorylation/dephosphorylation of specific residues on the GPCRs. For example, dephosphorylation of threonine residue (T353/354) on the somatostatin 2A receptor occurs at the plasma membrane independent of receptor internalization, but the dephosphorylation of serine residue (S341/343) happens post internalization [10, 112]. Similarly, internalization is required for dephosphorylation of histamine H₂ receptor for resensitization [113], and is supposed to be the norm in GPCR resensitization.

The above described reports, though limited, clearly suggest that resensitization as an independent process is tightly regulated and indepth studies are required to provide better understanding of this phenomenon. A key issue in resensitization is to determine the mechanisms regulating PP2A which mediates dephosphorylation of the receptors. Our recent studies have started to mechanistically address the regulation of PP2A wherein we have shown that activity of PP2A is acutely regulated [9]. Agonist activation of β-adrenergic receptors leads to recruitment of PI3Kγ to the receptor complex [63] that inhibits PP2A activity. Genetic or pharmacologic inhibition of PI3Kγ results in significant elevation of PP2A activity leading to consequential β-adrenergic receptor resensitization despite the agonist [9]. Uniquely, PP2A inhibition is mediated by protein kinase activity of PI3Kγ at the β-adrenergic receptor complex. PI3Kγ phosphorylates an endogenous inhibitor of PP2A (I2PP2A). Phosphorylation of I2PP2A by PI3Kγ significantly increases its affinity towards PP2A leading to enhanced binding of I2PP2A to PP2A. Elevated binding of I2PP2A to PP2A results in marked inhibition of PP2A activity [9] following agonist activation resulting in blockage of plasma membrane receptor dephosphorylation and subsequent resensitization. Consistent with this postulation, inhibition of PI3Kγ in vitro or in vivo results in plasma membrane resensitization of β-adrenergic receptors accounting for preservation of receptor function despite the presence of agonist [9]. These studies elucidate the presence of a PI3Kγ-I2PP2A-PP2A axis regulating receptor resensitization that is agonist dependent and step wise disruption of this axis results in plasma membrane β-adrenergic receptor resensitization [9].

PI3Kγ seems to play a critical role in dynamically shifting the receptor towards desensitization. PI3Kγ by its lipid/protein kinase activity increases β-adrenergic receptor internalization [62, 63] and by its protein kinase activity inhibits PP2A activity enhancing desensitization of the receptor [9]. This dynamic shift in equilibrium results in internalization of the receptor complex, (containing the receptor, PI3Kγ, PP2A, and I2PP2A) which is targeted into the endosomes. It is understood that these endosomes undergo acidification leading to activation of PP2A that mediates receptor dephosphorylation [101]. We have shown that acidification mediated by the H⁺ ATPase (V-ATPase) proton pump is a critical determinant of PP2A activity and receptor dephosphorylation. Blockage of the proton pump by bafilomycin inhibits PP2A activity and subsequent receptor dephosphorylation [9]. In this context, inhibition of PI3Kγ is sufficient to induce β-adrenergic receptor resensitization despite bafilomycin inhibition of the proton pump suggesting that PI3Kγ is the key molecule regulated by acidification. Importantly, significant loss of PI3Kγ from the endosomes is observed with concomitant acidification of the endosomes. Thus, loss of PI3Kγ from the endosomes results in increase of PP2A activity mediating dephosphorylation of the receptor leading to resensitization [9]. Consistent with these observations, the involvement of PI3K is implicated in P2Y purinergic receptor resensitization [114, 115]. The role of PI3K in resensitization is increasingly evident by defects caused by PI3K inhibitors in postendocytic sorting of β₂-adrenergic receptors [116].

The experimental evidence suggests that GPCR desensitization and resensitization is a seamless process that is tightly regulated by multiple players. Importantly, agonist activation of GPCRs results in measurable desensitization followed by accurate resensitization ensuing long standing responsiveness in the cells. In the context of resensitization, recent studies including ours suggest that recruitment of molecules to the receptor complex changes the receptor dynamics towards desensitization. Since these steps are dynamic in nature, it is potentially possible to reverse them by altering the function of critical proteins at certain stages in this process. Such a possibility opens up the option for resensitization to occur at the plasma membrane in an event when certain molecules that are critical to tilt the dynamics are not recruited to the receptor complex. We believe that one such molecule is PI3Kγ, as our data shows that inhibiting PI3Kγ results in plasma membrane receptor resensitization. Blocking acidification results in PI3Kγ being associated with high affinity towards receptor complex in the endosomes inhibiting PP2A activity [9]. Inhibition of PI3Kγ rescues PP2A function showing that primary effect of acidification is on PI3Kγ which then affects PI3Kγ-mediated regulation of PP2A activity [9]. Consistent with the observation that acidification is required to remove the PI3Kγ brake on PP2A activity, inhibition of PI3Kγ at the plasma membrane results in increased PP2A activity and resensitization of the receptors [9]. These data clearly provide the explanation and the reconciliation for the plasma membrane resensitization that could occur in the absence of PI3Kγ contrasting it with the occurrence of resensitization in the recycling acidic endosomes in the presence of active PI3Kγ. Presence of active PP2A at the plasma membrane should not be surprising as PP2A activity is observed in the cytoplasm and membranes where the surrounding environment is not acidic [7, 108]. This phenomenon of active plasma membrane PP2A activity can, to a certain extent, explain the pharmacologically observed transitions of the receptors from baseline to activated state and to baseline (R to R* to R) in the absence of agonists [31]. Based on our studies on plasma membrane PP2A activity [9], we postulate that receptors in activated states may actively be transitioned to baseline by PP2A activity thereby maintaining a dynamic equilibrium. The idea that PP2A is active at the plasma membrane and can bring about plasma membrane resensitization is supported by studies on neurokinin 1 receptor [7, 117]. Adding strength to this train of thought are the studies on μ-opioid receptors showing that resensitization is not blocked despite blocking μ-opioid receptor endocytosis [118, 119]. Together these studies have spurred the debate on the occurrence of both desensitization and resensitization at the plasma membrane [8, 10, 11], indicating that resensitization as a process could be independent of receptor internalization (Fig. 2). More indepth studies are required with regards to the location and function of PP2A to better integrate the coordinates of receptor resensitization. Based on the increasing evidence that resensitization could be as important a partner in receptor function as desensitization, better insights into the location and molecular mechanisms regulating GPCR resensitization is required which likely will identify targets for the development of novel therapeutic approaches.

Fig. (2). Shifting paradigm in GPCR resensitization.

Activation of GPCR by an agonist leads to the recruitment of GRK and PI3K to the receptor complex at the plasma membrane. While GRK phosphorylates the C-terminal tail of the receptor, PI3K inhibits the activity of the receptor dephosphorylating enzyme PP2A. PI3K phosphorylates an endogenous inhibitor of PP2A (I2PP2A) resulting in increased binding of I2PP2A to PP2A reducing latter's activity. Pharmacological or genetic inhibition of PI3K manifests in higher PP2A activity and rapid dephosphorylation of the receptors at the plasma membrane. Several recent studies suggest that GPCRs can be efficiently dephosphorylated and resensitized at the plasma membrane without necessarily undergoing internalization.

A question that has not been discussed till now is what happens to the agonist that is bound to the receptor in the acidic endosomes. It is believed that dephosphorylation may potentially cause a conformational change in the GPCR releasing the agonist. This is supported by recycling of the somatostatin receptor agonists back to the extracellular medium [120]. Indeed, inactivation resistant agonist of bradykinin B2 receptors revealed long lasting assembly of receptor/β-arrestin complex [121]. Similarly, differential receptor/β-arrestin complexes were observed based on the affinity of different chemokine ligands to the same CC Chemokine Receptor 2b showing that the ligand affinity plays a critical role in maintaining sensitization of the GPCR [122]. These studies, though few and far apart, provide pivotal insights regarding the fate of the agonist which will vary for each receptor based on the sensitivity of the agonist to degradation post internalization. These observations do warrant more studies in this area to better understand the fate of agonists after receptor internalization and its role in GPCR resensitization. Importantly the ability to generate inactivation resistant agonists provides exciting possibilities of novel therapeutic approaches.

IMPLICATION OF GPCR RESENSITIZATION IN PATHOLOGY

With increasing appreciation that resensitization is as important as desensitization, and an independent process in regulating GPCR function, it becomes imperative to assess whether alterations in resensitization could underlie pathological manifestation. Significant investment has been done in understanding desensitization and its contribution towards pathology with little efforts on resensitization. Accumulating evidence indicates that there is more to resensitization than a passive homeostasis maintaining process [117, 123-125] and thus may have significant contribution in pathologies wherein GPCR dysfunction is an underlying cause. Since it is known that GPCRs regulate a wide variety of responses including vision, olfaction, cardiac function, gestation, and neuro-transmission, it becomes essential to determine whether resensitization is altered in these pathologies. For example, it is well known that airway hyper-responsiveness in asthma is associated with β₂-adrenergic receptor dysfunction and one of the current treatment modality involves use of long acting β-agonist. Use of long acting β-agonist quickly results in the loss of efficacy for the control of asthma which may be due to the inability of β₂-adrenergic receptors to resensitize. Thus, if we had the indepth knowledge on mechanisms of resensitization like we have on desensitization, it would have provided us with novel therapeutic tools that could afford some respite to the patients. Similarly, β-adrenergic receptor desensitization is a major underlying cause in cardiac hypertrophy and failure [126]. β-adrenergic receptors accumulate in the endosomes during end-stage human heart failure [127], but it is not known whether these receptors are stranded in the endosomes due to loss or alterations in resensitization. These two examples are sufficient to exemplify the fact that we know very little about resensitization in pathology and it is potentially possible that deleterious alterations in resensitization may also be an active contributor.

Considering that resensitization is as important a step as desensitization in regulating GPCR function, we believe it is time to invest resources to determine comprehensively the mechanisms regulating resensitization. This is essential given the knowledge gap and the benefits we could reap by understanding resensitization. The benefits would be in the form of novel therapeutic strategies as the majority of current therapeutic targets have been myopically viewed through the lens of desensitization. Targeting resensitization per se opens up exciting possibilities as it now allows for altering GPCR function in unique ways. For example, GPCR ligands are used for pain, anxiety, depression, and neuroprotection, and their clinical use is limited due to development of tolerance upon long term administration [128, 129]. This tolerance or desensitization of receptors could potentially be overcome by activating resensitization as it is possible that resensitization is chronically blocked following prolonged presence of excess agonist. The finding that the morphine receptor ligand herkinorin can efficiently engage morphine receptor to G-protein coupling without initiating β-arrestin translocation despite GRK 2 overexpression [130], brings out exciting possibilities on designing opioid agonists that could prevent impairment of resensitization limiting tolerance [119]. These unique opportunities now provide an open field for development of next generation drugs that would target novel pathways. These exciting possibilities will translate into tangible targets only with better understanding of resensitization as a phenomenon that can independently regulate GPCR function. In this regard, the splice variant of cholecystokinin receptor 2, CCK2i4svR has rapid rates of resensitization which is believed to contribute towards progression and/or spread of colorectal and pancreatic cancer [125]. The above discussion highlights the urgency to understand the mechanisms regulating resensitization due to its ever increasing foot print in disease states.

CONCLUSION AND PERSPECTIVES

In this review we have tried to provide an overview on GPCR resensitization in the backdrop of our indepth and comprehensive knowledge on mechanisms regulating receptor desensitization. Our discussion on resensitization provides an idea to the readers that there are significant knowledge gaps due to paucity of available experimental data and observations to support the mechanistic understanding of resensitization. We believe that the time is ripe to appreciate that resensitization is not a passive homeostasis phenomenon but a regulated process which could independently alter GPCR function. In spite of conceptual support to the idea, that resensitization may be altered in human pathologies; little in terms of resources has been invested in understanding the mechanisms. Based on the discussion that we have presented and the knowledge that GPCRs are currently the major targets, it is time to have a fresh look at resensitization as a major regulatory mechanism of GPCR function. Thus, unraveling mechanistic details of GPCR resensitization will be a key step in developing novel therapeutic strategies and next generation drugs. More importantly, understanding resensitization opens exciting possibilities to generate biased agonists that would allow for receptor signaling without inhibiting receptor-associated phosphatase activity which may overcome tolerance. We hope that the review will provide a platform to promote active discussion regarding resensitization as an independent process regulating receptor function and value resensitization as an equal partner to desensitization.

ABBREVIATIONS

ADP

adenosine-5’- diphosphate

AKAP

A-kinase anchoring protein

AP-2

adaptor proein 2

ARNO

ADP-ribosylation factor nucleotide binding site opener

ATP

adenosine-5’-trphosphate

CCK

cholecystokinin receptor

CXCR

chemokine receptor

ERK

extracellular-signal regulated kinase

GDP

guanosine-5’- diphosphate

GIT

GRK interacting protein

GPCR

G-protein coupled receptor

GRK

G-protein coupled receptor kinase

GTP

guanosine-5’- triphosphate

I2PP2A

inhibitor 2 of PP2A

MAGI

membrane-associated guanylate kinase inverted

PDZ

acronym combining the first letters of three proteins — Post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and Zonula occludens-1 protein (zo-1)

PI3K

phosphoinositide 3-kinase

PP2A

protein phosphatase 2A

PSD

post synaptic density protein

siRNA

small interfering ribonucleic acid

USP

ubiquitin specific proteases