I. Introduction

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In the past two decades, a plethora of information has accumulated on -adrenoceptor (AR)² functional expression in both normal cardiac physiology and disease. The paradigm of this expression is that catecholamine binding to the -AR on the cell surface initiates a cascade of intracellular biochemical and molecular responses, leading ultimately to the stimulation of a number of cellular activities. At the onset, the activation of the -ARs directly triggers interactions of guanine nucleotide-binding proteins (G proteins) with adenylyl cyclases (ACs) to synthesize the second messenger cAMP from ATP (Gilman, 1987, 1990). The cascade of events progresses as cAMP in turn activates certain serine (Ser)/threonine (Thr) protein kinases (PKs), particularly protein kinase A (PKA), resulting in either an enhancement or inhibition of certain cellular functions. The ensuing responses are mediated by alterations in gene expression induced by the phosphorylation of specific transcription factors (Gilman, 1990; Hadcock and Malbon, 1991). The -AR-positive inotropism involves cAMP-mediated increases in intracellular Ca²⁺ concentration, resulting in part from the phosphorylation of L-type Ca²⁺ channels by PKA (Sperelakis et al., 1994; Hove-Madsen et al., 1996). This receptor system probably is the most extensively studied and best understood signaling pathway to date, yet it continues to attract great research interest, particularly with regard to its mode of signaling in cardiac disease. This is attributable mainly to the fact that in humans, although there are several receptor signaling systems capable of exerting positive inotropic effects via various mechanisms, the -AR pathway remains the most powerful tool by which heart rate and contractility are physiologically regulated and maintained (Brodde et al., 1992). On the other hand, the human heart possesses only a few spare receptors for -AR-mediated positive inotropy, requiring the mobilization of virtually all available cardiac -ARs to produce maximal inotropic effects at all times (Brodde et al., 1992). This phenomenon becomes particularly important in heart failure, where ₁-ARs are desensitized, often accompanied by the uncoupling of the ₂-AR subtypes (Bristow et al., 1986; Brodde, 1991). This scenario implies, among others, that any alteration in the cardiac contractile machinery capable of triggering down-regulation of -AR will automatically lead to a malfunction of this signaling pathway and is likely to prove detrimental to the cardiac circulatory function. Naturally, by virtue of its pivotal role to sustain life, the heart should have at its disposal the ability to regulate its receptor signaling pathways in a fashion that ensures continuity of this vital function, if and when the -AR system should fail. Several receptor systems, including the ₁-ARs, probably are capable of providing a compensatory mechanism against loss of the ₁-AR-positive inotropism in heart failure. However, although this notion has been entertained for almost a decade, no compelling evidence has come to surface to substantiate it. A further point of interest is the likelihood that the prevalence of certain genotypes and inherent genetic defects in some signaling components of the -AR pathway are potential markers for cardiac disease. This notion only adds a twist to the already complex and challenging task of defining the actual extent to which -AR signaling is involved in cardiac function in both the healthy and disease states. Even more exciting are data suggestive of a cross-talk at various signaling levels among the cardiac receptors, such as the renin-angiotensin-aldosterone (RAS) and atrial natriuretic peptide (ANP) systems, that contribute to cardiac circulatory function as well as receptors for growth factor systems. Such a cross-talk probably constitutes an integral regulatory network of the cardiac circulatory signaling complex, the essence of which is likely to be more eminent in cardiac disease than in normal physiological signaling. Our knowledge on this subject promises to increase exponentially in the foreseeable future. Indeed, the functional expression of the cardiac -AR pathway will soon be inconceivable without intimate linkage with other signaling systems contributing to cardiac circulatory function. This review thrives to integrate the current concepts on -AR signaling with the view of forging a basis to recognize this system primarily as an integral component of the complex machinery regulating cardiac circulatory function rather than an isolated regulator of the cardiac contractile apparatus.

	II. The Cardiac -Adrenoceptor Signaling Pathway

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The -ARs form part of a large superfamily of G protein-coupled, heptahelical, membrane localized receptors for drugs, neurotransmitters, and hormones. These G protein-coupled receptors (GPCRs) activate a small but diverse subset of effectors, including ACs, phospholipases, and various ion channels (reviewed in Karoor et al., 1996; Gudermann et al., 1997). Their expression is highly regulated and controlled largely by the activation or repression of genes encoding receptors, balanced by post-transcriptional mechanisms, such as the destabilization of receptor mRNA (Hadcock and Malbon, 1988). The -AR family transduces catecholamine signals by coupling to the large G proteins composed of G heterotrimers.³ In the basal state, the heterotrimeric G proteins have GDP bound to the catalytic site of GTPase on their G subunit. After interactions with the receptor, their activation requires association of GTP to the G in exchange for GDP, leading to the dissociation of the complex into GTP G and subunits. The dissociated G and G subunits then either positively or negatively regulate a variety of effector systems, resulting in changes in intracellular second messenger level and/or conductance (Northup et al., 1983; Birnbaumer, 1992). Hydrolysis of GTP to GDP by the complex results in the reassociation of the G with the subunits to commence the next activation cycle (Helper and Gilman, 1992). The affinity of the GDP- and GTP-binding sites of the G subunit varies with the occupation of the receptor, and both the G and complexes are known to determine the G protein specificity. The classic route leading to stimulation of the contractile function results from the activation of AC catalytic activity by the stimulatory G protein (G_s), leading to increased intracellular cAMP levels (Birnbaumer, 1992). This in turn stimulates PKA, which then mediates phosphorylation of target proteins such as the cardiac Ca²⁺ channels leading to various metabolic and physiological responses in the cells. Apart from this classic pathway, some evidence suggests that the heart may use other pathways in regulating its Ca²⁺ metabolism and therefore its contractile function by coupling -AR to G_q or G_i-G_o (Xiao and Lakatta, 1993; Zhou et al., 1997).

At least three human genes that express the individual -AR subtypes ₁-, ₂-, and ₃-AR have been identified so far, and unequivocal evidence for the existence of a putative fourth ₄-AR awaits its cloning and sequencing. The three known genes are characterized by an extracellular glycosylated amino (N) terminus, an intracellular carboxyl (C)-terminal region, and seven transmembrane domains (TDs) linked by three extracellular and three intracellular loops. The ₁- and ₂-ARs show 48.9% homology, whereas ₃ exhibits 50.7 and 45.5% homology with the other two receptors, respectively (Emorine et al., 1989). Several amino acids are conserved in all three proteins (Dohlman et al., 1987). Valuable information on the -AR functional domains, and indeed those of GPCRs in general, has been derived mainly from studies using the prototypic ₂-AR involving mutant receptors in which certain amino acids or regions were deleted or substituted, as well as synthetic chimeric receptors subtypes. Neither of the two receptor termini nor the hydrophilic loops are essential for ligand binding (Dixon et al., 1987a,b); rather, the catecholamine binding domain is a pocket lined by residues belonging to the hydrophobic TDs, which are also essential for protein folding involved in receptor-ligand interactions (Tota et al., 1991). The amino acid residues essential for agonist binding are different from those that are important in their interactions with antagonist (Dohlman et al., 1987; Tota et al., 1991). In particular, the Ser²⁰⁴ is presumably responsible for the receptor interactions with the parahydroxyl groups of the catecholamine ligands (Tota et al., 1991) and may be relevant for agonist high-affinity binding. Furthermore, the Asn²⁹³ has been associated with both stereospecificity and intrinsic activity of agonists in their interactions with the receptor (Wieland et al., 1996). For antagonists, on the other hand, the regions VI and VII seem to be essential in determining their specificity (Frielle et al., 1988; Kobilka et al., 1988). This differentiation in residues responsible for agonist and antagonist binding may provide a basis for delineating the specificity of ligand binding to -AR (Kikkawa et al., 1997, 1998). The region responsible for receptor interactions with the G_s protein is composed of residues belonging to parts most proximal to the membrane side of the third intracellular hydrophobic loop and C-terminal region (Strader et al., 1987; O'Dowd et al., 1988; Cheung et al., 1989; Strosberg, 1995). The first loop may be important for receptor expression, and Asp¹³⁰ in loop 2 is probably involved in the G_s coupling.

All four -AR subtypes are integral membrane proteins present in the human heart (Bylund et al., 1998). The ₁-AR is a protein of 477 amino acids found on chromosome 10 (Frielle et al., 1987), and it is distributed in all parts of the heart (Brodde, 1991). Stimulation of the cardiac ₁-AR leads to an increase in automaticity, conduction velocity (chronotropy), excitability, and contraction force (inotropy) of the cardiac muscle (Kaumann et al., 1989; Bristow et al., 1990). In the nonfailing myocardium, the ₁-AR population mediates the majority of the tensile responses to nonselective agonists (Brodde, 1991). The ₂-AR subtype consists of 413 amino acids and is located on chromosome 5 (Kobilka et al., 1987). It is concentrated mainly in the ventricles and atria, where it is similarly coupled to the myocardial contractile system (Bristow et al., 1986). In these tissues, the human ₂-ARs are also functionally linked to the cardiac positive inotropic responses to agonists (Summers et al., 1989). In both atria and ventricles, the ₁- and ₂-subtypes exist in a ratio of approximately 2:1. High proportions of the ₂-AR are apparently also found in the pacemaker and conducting regions, where they may be important in controlling heart rate and rhythm. The ₃-AR, on the other hand, is found mainly in the coronary vascular bed (Strosberg, 1995). It is 402 amino acids long and is located on chromosome 8 (Emorine et al., 1989). Although the cardiac ₃-AR is capable of exerting positive inotropic effects in isolated atria (Emorine et al., 1994), its actual contribution to the cardiac contractile function has yet to be defined more precisely. Recent studies have shown that the pharmacology of the human ₃-AR differs significantly from that of the other two human subtypes as well as the ₃-AR found in other species (Kaumann and Molenaar, 1996). The most striking difference is its recognition as agonists, several compounds acting as potent ₁-AR and ₂-AR antagonists, and its down-regulation by derivatives of various compounds, such as dexamethason butyrate or insulin, which up-regulate the other two subtypes (Silence et al., 1993). Hence, the existence of ₃-AR in the human heart initially led to the speculation that it may be responsible for the unexpected negative inotropic effects of catecholamines and that it may be involved in the pathophysiological mechanisms leading to heart failure (Gauthier et al., 1996). Although the notion of a putative fourth ₄-AR is about 20 years old, information on its pharmacological properties is just beginning to emerge. The receptor has been shown to mediate positive inotropic and chronotropic effects in mammalian hearts (Kaumann and Molenaar, 1997; Molenaar et al., 1997). In humans and a variety of other species, this receptor apparently mediates cardiostimulant effects of nonconventional partial agonists (i.e., high-affinity ₁- and ₂-AR blockers that cause agonist effects at considerably higher concentrations than those that block the receptors; Sarsero et al., 1998). The receptor is expressed in several heart regions, including the sinoatrial node, atrium, and ventricle. It has also been reported to mediate increases in Ca²⁺ transients in ventricular myocytes and to induce arrhythmias, probably via a mechanism that is different from that of ₁-AR (Kaumann and Molenaar, 1997, Lowe et al., 1998). All four -ARs are involved in the regulation of energy expenditure and lipolysis (Lafontan et al., 1997). The ₄-AR mediates lipolysis like the ₃-AR by interacting with the nonconventional agonists. However, it is not activated by selective ₃-AR agonists (Kaumann and Molenaar, 1996) and maintains its function in the hearts of ₃-AR knockout mice (Kaumann et al., 1998).

All four -AR subtypes are coupled to their effector systems by G proteins (Bylund et al., 1998). The human ₁-, ₂-, and ₄-ARs are coupled mainly to the stimulatory G_s-AC system, whereas the ventricular ₃-AR is coupled to G_i (Kaumann and Molenaar, 1997). Interestingly, although most of the ₂-AR actions are mediated by the G_s protein coupling via the cAMP-dependent PKA system, at least three other signal transduction cascades have been described by which it can mediate various agonist effects. In 1993, Xiao and Lakatta described a ₂-AR-stimulated cAMP production that was dissociated from the regulation of myofilament and sarcoplasmic reticulum functions. Because ₂-AR modulation of cardiac excitation-contraction coupling requires cAMP, a functional compartmentalization of cAMP signaling was postulated that is due to the activation of ₂-AR coupled to G_i and/or G_o to explain this phenomenon (Zhou et al., 1997). There also is compelling evidence indicating that the PKA-mediated heterologous desensitization of the ₂-AR pathway may actually serve an independent physiological signaling route rather than simply an escape circuit for the receptor from unabated stimulation by agonists. This is based on a recent finding by Daaka et al. (1997a) describing a switching of the ₂-AR coupling, in which the phosphorylated and internalized ₂-ARs tend to lose affinity for G_s and gain affinity for G_i. Besides, there is some evidence to suggest that certain physiological functions of the ₂-AR do not depend entirely on G protein activation but may be a result of protein-protein interactions mediated by conserved protein modules known as PDZ domains (derived from the three proteins PSD-95, Dlg, and ZO-1). These are protein-recognition modules of approximately 90-residue repeats found in a number of proteins implicated in ion-channel and receptor clustering, and the linking of receptors to effector enzymes and are specific for the postsynaptic scaffolding protein of 95 kDa (PSD-95), Drosophila discs-large tumor-suppressor gene (Dlg), and zonula occludens protein (zo)-1. Hall et al. (1998) reported that ₂-AR controls the Na⁺/H⁺ exchange via its direct agonist-promoted association with the Na⁺/H⁺ exchanger regulatory factor (NHERF) type 3 as a result of the NHERF binding to the last C-terminal cytoplasmic residues of the receptor by means of PDZ domain-mediated interactions. These observations indicate that -AR signaling is finely tuned by an interplay of several regulatory mechanisms, notably at the G protein coupling level, and that further diversity is likely to surface soon, which should enhance our understanding of the different modes by which their regulation contributes to cardiac function.

The heterotrimeric G proteins are a family of proteins composed of , , and  subunits of 45 to 52, 35 to 37, and 8 to 10 kDa, respectively, that play a key regulatory role as transducers of various signaling pathways in different cell types (Gilman, 1987). To date, at least 20 G, 5 G, and 11 G subtypes of the G protein have been identified (Table 1). The G subunits differ significantly from one another, whereas the G and G subunits do not vary remarkably among the G proteins, with the G subunits exhibiting higher sequence similarities than the G group. Among the G proteins, the stimulatory G_s and inhibitory G_i are highly homologous, but they differ profoundly with respect to their effector, regulator, and receptor specificities. At least seven of the G proteins (four G_s gene splice variants and one each of G_i1, G_i2, and G_i3) are involved in the regulation of the AC signaling mechanisms. Particularly relevant for cardiac -AR signaling is the fact that G_s activates all AC isoforms, whereas the G_i proteins inhibit only certain isoforms, notably AC IV and V, depending on the nature of the enzyme activators (Tang and Gilman, 1991; Taussig et al., 1993). The G_s is abundantly expressed in the myocardium, whereas among the G_i subunits, G_i2 is predominant in the heart with a little of G_i3 (Luetje et al., 1988; Holmer et al., 1989). However, the relevance of not only their presence in the heart but also their diversity with regard to cardiac -AR signaling under physiological conditions remains somewhat elusive. Apparently, ₁-AR and ₂-AR share at least one coupling domain within the G_s for its activation (Novotny et al., 1996). The most significant role for the G_s coupling has been assigned to the third loop, which, among others, accommodates the sites involved in the guanylyl cyclase (GC) coupling (Summers and MacMartin, 1993). Basic amino acids, such as His²⁶⁹ and Lys²⁷⁰ in this region and Pro¹³⁸ in the III/IV segment, are thought to be particularly important for the receptor/G protein coupling (Strader et al., 1987).

It was not until recently that the G complex was recognized as a signal transducing molecule in its own right that directly regulates just as many different protein targets as the G. Apart from its differential regulation of the various AC isoforms (Tang and Gilman, 1991), the complex also directly stimulates several effector systems, including PLC, a cardiac potassium channel, a retinal PLA₂, and a specific receptor kinase (Clapham and Neer, 1997). Its synergistic enhancement of the GPCR-mediated activation of GRK2 has led to the proposition of a pleiotropic function in its regulation of GPCR signal transduction (Haga et al., 1994). Of the 12 G subunits, at least 5 (1, 2, 3, 5, and 7) are expressed in the rat heart (Hansen et al., 1995), displaying a huge potential for diversity in the coupling of the G protein subunits to transduce cardiac GPCR signaling in particular.

In cardiac myocytes, the Ca²⁺ required for the activation of the contractile proteins is furnished by the Ca²⁺ current (I_Ca) emitted via the slow (L-type) Ca²⁺ channel (Sperelakis et al., 1994). The availability of these channels for voltage activation during excitation is regulated, at least in part, by cAMP through both direct and indirect pathways. An elevation in cAMP levels produces a very rapid increase in the number of channels available for activation, thereby augmenting also the probability of a Ca²⁺ channel opening and its mean opening time. ACs constitute the effector enzymes that catalyze the conversion of ATP to cAMP, a ubiquitous second messenger that mediates diverse cellular responses primarily by activating Ser/Thr PKs. At least 10 mammalian AC isoforms have been identified so far, all of which are membrane-bound enzymes, with molecular masses of 115 to 150 kDa exhibiting overall homology of roughly 60% and shared identity of 50 to 90% within the two cytoplasmic regions (Krupinski et al., 1989; Iyengar, 1993; Sunahara et al., 1996). The cytosolic regions represent the catalytic sites of the ACs, exhibiting approximately 50% similarity and 25% identity (Sunahara et al., 1997). The isoforms share several regulatory features, including activation by the G_s and forskolin and inhibition by a class of adenosine analogs known as P-site inhibitors (Iyengar 1993). They appear to fall into three broad categories. The first group represents Ca²⁺-calmodulin (CaM)-stimulated enzymes that are activated synergistically by G_s and Ca²⁺-CaM (types AC I, III, and VIII). The second group consists of isoforms that are activated synergistically by G_s and G (types AC II, IV, and VII), and the third is composed of the isoforms that are inhibited by G_i and Ca²⁺ (types AC V and VI; Sunahara et al., 1996). The isoforms can also be individually regulated by Ca²⁺-CaM, Ca²⁺, or phosphorylation processes (Yu et al., 1990; Xia and Storm, 1997). Incidentally, AC V and VI, which are inhibited by Ca²⁺, are also distinctly expressed in excitable tissues, particularly in the heart and the brain. In cardiac myocytes, where AC V and VI are most abundant, inhibition of their catalytic activity results primarily from the activation of L-type Ca²⁺ channels (Iwami et al., 1995). The diversity in the way by which -AR signaling regulates Ca²⁺ metabolism is already evident at the G protein coupling level. At least three pathways for enhancing cAMP synthesis by -ARs have been delineated involving signaling coupling via the G_s, G_q, and G_i/G_o proteins, respectively. The G_s-coupled -AR signal transduction directly enhances cAMP synthesis. The G_q does so via Ca²⁺ and protein kinase C (PKC), whereas the G_i and G_o do so via the complex. On the other hand, the most obvious mechanism for the inhibition of cAMP synthesis in the heart probably is the direct interaction of G_i, notably G_i2, with AC after the activation of the G subunit and its dissociation from the complex. Moreover, the different AC isoforms are phosphorylated by different types of PKCs in a fashion that is synergistic to that of forskolin or G_s (Jacobowitz and Iyengar, 1994; Kawabe et al., 1994). AC V is a substrate for phosphorylation by PKC- and PKC- in vitro, whereas AC II and IV are phosphorylated in vivo in response to phorbol 12-myristate 13-acetate (PMA). This regulatory diversity allows each AC isoform to respond to intercellular and intracellular signals in a manner appropriate for the individual cellular and environmental requirements, which might be particularly important in receptor cross-talk in cardiac disease. This renders the ACs as important targets for direct or PKC-mediated modulatory effects of Ca²⁺ and therefore as essential nodes for integrating the crucial cAMP- and Ca²⁺-regulated signaling systems (Sunahara et al., 1997). Similarly, members of the G_q protein family also should be capable of modulating the AC function indirectly via their effects on phospholipase C (PLC)- and inositol-1,4,5-triphosphate (IP₃) synthesis by coupling to PKC. Furthermore, both Ca²⁺-mediated inhibition of the cardiac AC V and VI and inhibition of their phosphorylation by PKA may provide a suitable negative feedback loop for the events leading to cAMP-mediated increases in intracellular Ca²⁺ (Iwami et al., 1995; Tang and Gilman, 1995).

PKs are phosphotransferases that catalyze the transfer of the -phosphoryl group of ATP to an amino acid side chain in basic sequences in the presence of Mg²⁺ in their post-translational regulation of receptors and in directing the downstream trafficking of receptor-mediated signaling messages. The PKs involved in signal transduction are divided into two major classes: those that phosphorylate Ser/Thr and those that phosphorylate Tyr. PKC and PKA are the two important Ser/Thr PKs involved in -AR signal transduction mechanisms. PKA is a tetramer of two regulatory subunits, associated with catalytic subunits termed C, C, and C exhibiting different substrate and inhibitor specificity, as well as requiring different cAMP levels for activation (Tasken et al., 1997). In cardiac myocytes, this enzyme is found both in the cytosol and in association with the intracellular membrane, a fact that may be important with respect to the postulated compartmentalization of cAMP-mediated signal transduction discussed below. PKCs, on the other hand, comprise a family of at least 12 isoforms of Ca²⁺- and phospholipid-dependent kinases encoded by nine genes (Nishizuka, 1988; Asaoka et al., 1992). They constitute key enzymes for growth regulation as well as tissue and cell differentiation and are extensively involved in cross-talk among cardiac receptor signaling pathways. Like PKA, the PKCs have four conserved domains (C1-C4). They are classified into three groups based on their structure and cofactor regulation. The best characterized are the conventional PKCs (cPKCs), which consist of the , two alternatively spliced  variants (I, II), and the  isoenzymes. This group distinguishes itself from the others in that its C2 domain contains a putative binding site for Ca²⁺ that regulates its function. The second class are the novel PKCs (nPKCs) , , (L), , and µ isoenzymes, which are structurally similar to the cPKCs, except that the C2 domain does not have the functional groups to mediate Ca²⁺ binding. The third class consists of the atypical PKCs (aPKCs) , , and  isoenzymes, which contain only one Cys-rich motif in C1 and lack the key residues that maintain the C2 fold. The heart contains a large amount of Ca²⁺-dependent isoforms , I, and II but not  or the Ca²⁺-independent isoforms  and  (Mochly-Rosen et al., 1990; Inoguchi et al., 1992). The relevance of this diversity and the Ca²⁺-independent isoforms for the regulation of cardiac function is not yet fully understood. However, there is mounting evidence showing that both PKA and PKC act as hubs for sorting adrenoceptor signaling routes involving cross-talk with other cardiac signaling systems as well as tyrosine receptor kinase pathway to stimulate the mitogenesis (Fig. 1).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Regulation of the Ras/Raf/MAP kinase pathway by adrenoceptors. Catecholamine stimulation of 1-AR activates this system via the cAMP-PKA, and stimulation of 1-AR activates this system via the 1,2-diacylglycerol/IP3-PKC pathway. The 2-AR can use both classic pathways. Both PKC and PKA serve as hubs for the cross-regulation of the adrenoceptors, but cross-talk may occur already at the second messenger levels. The PKA-mediated 2-AR desensitization pathway may also stimulate the Ras/Raf/MAP kinase pathway by coupling to Gi/Go via Src/Sos pathway. The various PK isoforms may act specifically to sort and sieve the individual signaling messages.

Protein phosphatases (PPs) are phosphotransferases that catalyze the transfer of phosphoryl groups from the phosphorylated side chain of an amino acid to water molecules, thereby controlling a variety of physiological events such as cell proliferation, cell cycle, and receptor recycling. Unlike PKs, the PPs belong to several protein and/or gene families. Three prominent types of phosphatase catalytic subunits have been identified, termed PP-1, PP-2A, and PP-2B based on their substrate preferences, mechanisms of activation, and sensitivity to inhibitor proteins or naturally occurring toxins (Faux and Scott, 1996). The regulation of PPs appears to involve protein-protein interactions controlled by phosphorylation and to be independent of second messengers (Girault, 1993; Hubbard and Cohen, 1993). Several Ser/Thr phosphatases and kinases are associated with targeting subunits that contribute to the organization and specificity of signal transduction pathways by favoring the accessibility of their enzymes to certain substrate proteins (Faux and Scott, 1996). For this purpose, compartmentalization has been suggested as a means to attain some measure of selectivity toward the physiological substrates at the plasma membrane and is thought to be particularly important with respect to the function of PKs and PPs (Newton, 1995).

In 1986, Lefkowitz and associates identified a cAMP-independent kinase that specifically phosphorylates the agonist-occupied form of the -ARs, which they called -adrenergic receptor kinase (ARK1; Benovic et al., 1986). This discovery was immediately followed by the realization that ARK1 was only a member of a family of proteins that specifically phosphorylate GPCRs leading to their desensitization (Benovic et al., 1989). Hence, these proteins are now more commonly known as the G protein-coupled receptor kinases (GRKs). At least six members of this family (GRK1-6) sharing 53 to 93% overall sequence homology have been identified and sequenced to date, and more may be in the pipeline (Premont et al., 1995). They have been subdivided into three groups according to their sequence homology and functional similarities. Group 1 consists of the rhodopsin kinase (GRK1), which is predominantly localized to the retina. Group 2 consists of the -AR kinase (referred to as either ARK1 or GRK2) and ARK2 (also known as GRK3), which exhibit a more ubiquitous tissue distribution, and the third subfamily is composed of GRK4, GRK5, and GRK6 (Premont et al., 1995). GRK4 is localized primarily to the testis, whereas GRK5 and GRK6 are more ubiquitously expressed. The common structure of GRKs consists of three main domains. One of these is a centrally localized highly conserved catalytic domain of approximately 240 amino acids, which shares significant amino acid identity (46-95%). This domain is flanked by a conserved N-terminal sequence of 161 to 197 amino acids (except for the Drosophilia kinase 2) and a variable length of C-terminal domain of 100 to 263 amino acids (Benovic and Gomez, 1993). The N-terminal domain is considered important for the recognition of activated receptor substrates, whereas the C-terminal domain probably is important for their membrane targeting (Palczewski et al., 1992) and harbors the autophosphorylation sites of GRK1 and GRK5 and the putative sites of GRK4 (Premont et al., 1996).

The most explicit functional role of GRKs is their preferential phosphorylation of several Ser/Thr residues of an agonist-occupied GPCR in either the C-terminal tail (e.g., rhodopsin and ₂-AR) or the third intracellular loop of the receptor (e.g., M₂ muscarinic acetylcholine receptor) in an agonist-dependent manner (Fredericks et al., 1996). Several GPCRs have been shown to act as substrates both in vitro and in vivo for various GRKs. Notably, although GRK2 is capable of using primarily the ₂-AR and muscarinic receptors and, to lesser extent, rhodopsin (Kwatra et al., 1989; Roth et al., 1991; Pippig et al., 1993), it prefers -AR as substrate to the other receptors (Lohse et al., 1990; Müller et al., 1997). For the GRKs to mediate phosphorylation of their substrates, they have to be associated with the plasma membrane. The different subfamilies use different mechanisms to achieve this. The first subfamily, GRK1, GRK2, and GRK3 which are primarily cytosolic proteins, translocate to the plasma membrane on receptor stimulation (Strasser et al., 1986; Inglese et al., 1993; Freedman and Lefkowitz, 1996). In contrast, GRK4, GRK5, and GRK6 are not isoprenylated, do not bind to G, and do not show agonist-dependent membrane association (Premont et al., 1996). Despite these differences among the three subfamilies, the membrane association of all of them appears to be mediated at least in part by residues in their C-terminal domains, such as the CAAX motif of the GRK1 (Inglese et al., 1992). This motif is supposed to direct its isoprenylation and caboxymethylation, whereas protein-protein interactions of the G complex probably take place with the C-terminal Pleckstrin homology domain of GRK2 (Koch et al., 1993; Müller et al., 1993; Parruti et al., 1993). For the first group, isoprenylation is believed to play a central role in mediating their membrane association either through direct covalent modification of the kinase (GRK1) or through protein-protein interactions between the kinase and the isoprenylated G (GRK2 and GRK3). It is postulated that after the activation of the G protein and its dissociation into G and G, the latter subsequently interacts with the GRK and serves to target these enzymes to their membrane-incorporated substrates (Daaka et al., 1997b). Although it is generally accepted that G stimulates GRK2/3, the exact mechanism of these actions has yet to be elucidated. Pitcher et al. (1992) originally proposed that the G primarily translocates the GRK from the cytosol to the plasma membrane to permit GPCR phosphorylation. In contrast, Inglese et al. (1992) suggested that the GRK binds to G to access its isoprenyl group for the membrane localization because it lacks this group in its C terminus. Besides GRK2 translocation, the G is associated with an additional role of facilitating its interactions with the activated receptor substrates resulting in the allosteric activation of the kinase (Kim et al., 1993; Haga et al., 1994). Unlike the members of the first subfamily, which engage isoprenylation in their membrane association, GRK4 and 6 probably use palmitoylation for this purpose (Stoffel et al., 1994; Premont et al., 1996). Site-directed mutagenesis studies have revealed a cluster of three Cys residues (Cys⁵⁶¹, Cys⁵⁶², and Cys⁵⁶⁵) in the C terminus of the GRK6 as a region of palmitate attachments, consistent with the location of membrane targeting domains of GRK1, GRK2, GRK3, and GRK5 (Stoffel et al., 1994). The palmitoylation of these Cys residues may function as a link between its activity toward its receptor substrates and its membrane association (Loudon and Benovic, 1997). By analogy with GRK6, the C-terminal Cys⁵⁶¹ and Cys⁵⁷⁸ of GRK4 are the probable sites of its palmitoylation (Stoffel et al., 1997). GRK5, on the other hand, is apparently constitutively associated with the membrane (Premont et al., 1995, 1996), an observation that might explain its higher basal activity compared with GRK2, for example (Premont et al., 1994). Two distinct lipid-binding domains of GRK5 have been identified, one in the N terminus and another in the C terminus, that may be involved in its receptor association (Stoffel et al., 1997). Besides, all five nonisoprenylated GRKs seem to require the presence of lipid cofactors for their interactions with their receptor substrates (Stoffel et al., 1997). Both the enhancement of GRK-mediated GPCR phosphorylation, by targeting the GRK to the membrane and locating the enzyme into close proximity with its receptor substrate, and the direct enhancement of GRK catalytic activity have been associated with the lipid-dependent nature of the GRKs. Lipids such as phosphatidylinositol-4,5-bisphosphate (PIP₂) have been shown to promote GRK2/G complex formation by binding to the N terminus of GRK2 (DebBurman et al., 1996). This led to the notion that the coordinated binding of PIP₂ to this N terminus and G to the C-terminal Pleckstrin homology is necessary for its effective membrane localization and function. In contrast, negatively charged phospholipids such as phosphatidylserine enhance GRK2-mediated -AR phosphorylation, presumably as a direct result of increasing the GRK2 catalytic activity (DebBurman et al., 1995). Therefore, the receptor regions responsible for GRK activation are probably distinct from the sites of GRK-mediated phosphorylation. The current view is that GRKs not only phosphorylate, bind, and uncouple the agonist-activated receptor but also play a pleiotropic role in their regulation of receptor responsiveness by providing the signal and serving as the molecular intermediates directing the agonist-promoted sequestration of the ₂-AR (Ferguson et al., 1997). Also noteworthy is the recent observation that PKC-mediated phosphorylation of certain C-terminal residues of GRK2 not only up-regulates its own activity but also may influence its targeting to the plasma membrane, implicating PKC as a direct regulator of GRK-mediated receptor desensitization (Winstel et al., 1996). Therefore, apart from communicating with each other via the normal physiological route, these two kinases may do so under conditions leading to -AR desensitization. Furthermore, the GRK and PKC are engaged in cross-talk in their activation of several receptor systems, notably those that are involved in growth regulatory mechanisms, adding yet another twist to our understanding of the role of phosphorylation in GPCR signaling, which should command great future research interest.

Soon after the discovery of GRKs, it became apparent that although homologous desensitization of GPCRs is triggered by their specific phosphorylation, this alone was insufficient to inhibit receptor function. It was realized that under physiological conditions, GRKs required the presence of a protein cofactor to accomplish receptor phosphorylation. The existence of such an arresting protein (visual arrestin) was postulated by Benovic et al. (1987) almost accidentally as a result of their experiments demonstrating the binding of a 48-kDa soluble retinal protein to GRK2-phosphorylated rhodopsin. The observations from this study led the investigators to suggest that a protein analogous to retinal arrestin may exist in other tissues and function in concert with GRK2 to regulate the activity of AC-coupled receptors. This finding was succeeded by experiments of Lohse et al. (1990) suggesting that appropriate homologous ₂-AR desensitization by GRK2 in vitro also required an "arrestin-like" protein (-arrestin). It was also shown that in Chinese hamster ovary cells expressing high levels of ₂-ARs, -arrestin, and GRK2 become limiting for homologous receptor desensitization, providing support for their involvement in the regulation of ₂-AR (Pippig et al., 1993). To date, six members of the family have been identified, and they exhibit 44 to 84% sequence homology: 1) visual arrestin (S antigen) localized primarily to the retina but also found in the pineal and primary blood leukocytes; 2) -arrestin-1 (bovine arrestin) and -arrestin-2, which are more ubiquitously expressed but most highly concentrated in nervous and lymphatic tissues; 3) cone arrestin (also known as C- or X-arrestin), which is found primarily in the cones but also is localized to the pineal, pituitary, and lung tissues; and 4) D-arrestin and E-arrestin, which are partial arrestin clones that have not been characterized but exhibit specific tissue distribution (Attramadal et al., 1992; Calabrese et al., 1994). Arrestins have several functional domains that contribute to the multisite binding of their receptor substrates: 1) a C-terminal acidic region that serves a regulatory role in controlling arrestin binding selectivity toward the phosphorylated and activated form of the receptor without participating in receptor interaction, 2) a basic N-terminal domain that directly participates in receptor interactions and serves a regulatory role via intramolecular interactions with the C-terminal acidic region, and 3) two centrally localized domains that are directly involved in determining receptor binding specificity and selectivity (Gurevich et al., 1995). Overall, -arrestin-2 exhibits 78% amino acid identity with -arrestin-1, both of which preferentially interact with GRK1, GRK2, and GRK5 in their regulation of -AR phosphorylation to other GPCRs as substrates (Attramadal et al., 1992; Freedman et al., 1995). Besides their apparently dual regulatory role in the GPCR life cycle in controlling both their desensitization and internalization, there are strong indications that both GRKs and arrestins might have a greater role to play in, for example, cardiac development or their regulation of the -AR signaling (Jaber et al., 1996; Rockman et al., 1998).

	III. The -Adrenoceptor Signaling Circuits

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The mechanisms by which GPCRs interact with their downstream effector molecules in transmitting signals of their primary messenger is only beginning to be deciphered, thanks to the arrival of molecular techniques that have allowed us to examine these individual processes at single-amino acid functional levels. These techniques have revealed that the interactions of GPCRs and their effector molecules are a dynamic process requiring various coordinated interactive cycles. Because these interactions take place in specific cellular compartments or organelles under stringent conditions, both the receptors and their downstream signaling components are subject to structural transformations and translocations by stringently coordinated mechanisms to ensure proper signal propagation. To initiate the signaling, the formation of the receptor active state may depend on agonist binding promoting a conformational change in the receptor, constituting a post-translational acylation by natural fatty acids in the form of palmitoylation, myristoylation, or prenylation (Rando, 1996; Stoffel et al., 1997). Also, the hormone-sensitive ACs have been localized to a specialized subdomain of the plasma membrane called caveolae that is believed to contain G proteins to optimize their signal transduction efficiency and specificity (Huang et al., 1997). This means that the cytosolic signaling components must be brought into close proximity with the plasma membrane-bound receptors and the AC. The G proteins also undergo several modifications, such as isoprenylation/methylation, possibly to enhance their association with the plasma membranes and therefore facilitate their signaling capability. Myristoylation of the N termini of the G proteins and/or binding to (and some other factors) probably directs them to a membrane location where palmitoylation takes place. The geranylgeranylation of the G subunits (or farnesylation as in the case of retinal G subunit) probably is required for correct targeting of the G dimer (Wedegaertner et al., 1995), whereas prenylation of the complex is thought to be a prerequisite for productive interactions of the complex with the G subunits, receptors, and effectors (Casey et al., 1994). On the other hand, the G subunits are predicted to form coiled-coil structures in their N-terminal region signifying stabilized  helices important for protein-protein interactions, especially with the G subunit. There also is some evidence to suggest that caveolins, the structural components of caveolae, play pivotal roles in the transportation of molecules and cellular signaling by interacting directly with and regulating the function of G proteins (Li et al., 1995). In the heart, the expression of caveolin subtypes is apparently regulated by -AR stimulation (Oka et al., 1997). However, there is much to be elucidated regarding the regulators of the mechanisms that switch on GPCR signaling.

The -AR-G protein-AC circuit consists of three independent but tightly interlinked signaling cycles: 1) the formation of a complex by the agonist-occupied receptor with the G protein and AC to initiate the signaling, 2) the G protein cycle to stimulate and regulate the coupling mechanism, and 3) the AC-G protein cycle regulating the AC catalytic function. Originally, a "ternary complex" model was suggested by De Lean et al. in 1980 to explain the agonist-specific binding properties of the AC-coupled -AR. In essence, this model suggests an agonist-promoted formation of high-affinity ternary complex composed of the hormone, receptor, and AC at the onset of the signal transduction, which is destabilized by the addition of a guanine nucleotide, leading to the dissociation of both the hormone and AC. The model was initially found to accurately fit data obtained with a full or partial agonist, situations in which a system was altered by the addition of a guanine nucleotide, or after treatment with agents specific for a particular group. It was then discovered that many GPCRs have the spontaneous capacity to activate their cognate G proteins and regulate downstream effectors in the absence of an agonist ligand and that designed mutational modifications of GPCRs known as constitutively active mutant (CAM) receptors could increase their extent of agonist-independent signal transduction (Lefkowitz et al., 1993; Samama et al., 1993, 1997). The CAM ₂-AR exhibits not only agonist-independent activation of AC but also increased affinity for agonists (even in the absence of G proteins) but not antagonists, whereby the increase in affinity correlates with the intrinsic activity of the ligand, including partial agonists (Samama et al., 1993). These authors noted that the ternary complex model previously suggested by De Lean and colleagues was not sufficient to adequately explain such phenomena; rather, an explicit isomerization of the receptor into an active state would conform to conditions for both the mutant and the wild-type receptors. Recently, it was reported that the mutation conferring constitutive activity to ₂-AR removes some stabilizing conformational constrains, allowing the CAM to undergo transitions more readily between the inactive and the active states and thereby making the receptor more susceptible to denaturing (Gether et al., 1997a). For the ₂-AR, the movements around Cys¹²⁵ in the TD III and Cys²⁸⁵ in TD VI are thought to be involved in this activation (Gether et al., 1997b). More recently, it was suggested that the association of, and equilibrium among agonist, antagonist, and G protein, as well as other factors within the membrane environment, consists of two events. First, an association of two domains, the TD helices 1 through 5 (domain I) and TD helices 6 and 7 (domain II), probably is necessary for signal transduction. Specific membrane perturbations of the conformational state of receptor side chains in the vicinity of the binding site of the agonist followed by G protein association may also be required for agonist activity (Underwood and Prendergast, 1997). However, it is still not known how and where in the cell such complexes are assembled and disassembled. Regardless of the way by which the -AR, G protein, and AC complexes are initiated, it is well established that the conformational change in the receptor induces the release of GDP in exchange for GTP on the G subunit, leading to the stimulation of AC catalytic activity. Two independent views are being entertained with respect to the activation of the AC by the -AR after the complex formation. One line of thinking purports that GPCRs govern their effectors indirectly via a two-step shuttling mechanism that involves the exchange of G proteins or their components between ephemeral GPCR-G protein and G protein-effector complexes. This may require the stimulation of AC by -AR involving, first, the activation of G_s independent of the cyclase, followed by G_s activation of the cyclase independent of the receptor. The second notion envisages the activation of a preoccupied G_s-AC complex by the receptor in a single step. Several experimental models have been put forward in favor of both mechanisms. Arguments for the shuttle mechanism are based on experiments pointing to changes in second messenger production, GTPase activity, and the binding characteristics of agonists, antagonists, and guanine nucleotides, as well as experimental evidence for the occurrence of the receptor-G protein-effector complexes. Although there is no direct documentation for the cyclical formation and dissociation of these complexes during signaling, it has been argued that they nevertheless reflect on perturbations in the equilibrium between the G protein and the other two components (Krumins et al., 1997). On the other hand, some investigators argue that the random, transient association of the G protein and the receptor is largely inconsistent with the binding of agonists to receptors and the allosteric regulation of that binding by guanine nucleotides. Furthermore, this paradigm does not readily account for receptor-effector coupling specificity, because the promiscuous interaction of most G proteins with both receptors and effectors in vitro is at odds with the general inability of the G proteins to be shared among highly congruous signal transduction pathways in vivo (Chidiac, 1998).

The cardiac-specific AC function is predominantly under the dual stimulatory and inhibitory regulation by receptors acting through the G_s and G_i proteins, respectively (Helper and Gilman, 1992; Spiegel et al., 1992; Eschenhagen, 1993). For the -AR-mediated stimulation of cAMP synthesis, the interaction between G_s and the two AC cytoplasmic domains (C1 and C2) constitutes a key step. This action is initiated by the dissociation of the complex as a result of the conformational changes after the catalysis of GDP-GTP exchange by the agonist occupation of the GPCR. Termination of the AC activation by G_s-GTP is rapidly achieved by the intrinsic GTPase activity of the G subunits initiating the hydrolysis of G_s-bound GTP to GDP. Mutation analysis reveals three discrete regions in the C2 primary sequence of AC that are close together but are distal to the catalytic site and probably function as the affinity determinants of the G_s protein. At present, a number of ideas are being entertained as potential mechanisms for the stimulation of AC activity by the G_s, including the possibilities that 1) the G_s directs the productive formation of a complex interface between the conserved units, 2) the G_s acts primarily as an allosteric activator of AC, and 3) it activates AC primarily by stabilizing a catalytically competent form of the enzyme (Tesmer et al., 1997). All of the residues that interact with G_s are conserved among AC isoforms (Yan et al., 1997). Only two of the G_s residues involved in the interfaces with AC, Gln²³⁶ and Asn²³⁹, are significantly different from the analogous residues His²¹³ and Glu²¹⁷ in the G_i subunit (Sunahara et al., 1996). The Phe³⁷⁹ in the conserved domains may be important for the activation of AC by the G proteins, whereas Arg⁴⁸⁴ probably is involved in G_s activation (Tang and Gilman, 1991; Taussig et al., 1994). The latter is a pyrophosphate-binding residue whose proximity to other residues in the active site may be essential for positioning other residues such as the Arg¹⁰²⁹ to interact with the perivalent transitional state of the substrate. By so doing, G_s can improve the stabilization of this transition state and potentiate a chemical step in the reaction mechanism (Tesmer et al., 1997). The binding site for the G_i is distinct from that of the G_s subunit. It is most likely situated between the 1-2 loop and the 3 helix on the C1 subunit directly opposite to the binding site of G_s. This region contains many residues such as Glu³⁹⁸ and Leu⁴⁷² that are invariant in AC V and VI but not in G_i-insensitive cyclases (Tesmer et al., 1997). These differences suggest that the specificity of G_s and G_i subunits for AC is dictated primarily by the backbone conformation of the residues that form the interface with the enzyme rather than their primary structure (Sunahara et al., 1996).

The mechanism by which the ACs catalyze the conversion of ATP to cAMP is still not fully understood. One line of thinking implicates its cytosolic conserved C1 and C2 domains singly or in combination as representing the AC catalytic sites for this function (Coleman et al., 1994; Zhang et al., 1997a,b). Their notion is based on the argument that the sequence homology of these AC domains is similar to GC domains believed to perform the same functions. A completely different school of thought advanced by Tesmer et al. (1997) suggests that both conserved domains provide the binding sites for ATP, and therefore the individual cytosolic domains are unlikely to be catalytic. Rather, a direct in-line attack of the O3'-hydroxyl on the 5'-phosphate of ATP without the formation of a phosphor-enzyme intermediate may account for the observation that AMP cyclization proceeds with an inversion of the configuration at the -phosphate (Tesmer et al., 1997).

The ultimate product of AC stimulation by the cardiac -AR pathway is the activation of the slow Ca²⁺ channel to regulate cardiac positive inotropy. This channel is a complex structure of several regulatory proteins whose function is controlled by a number of factors that are intrinsic and extrinsic to the cell. After its AC-mediated synthesis from ATP, cAMP regulates a series of cellular functions mediated by PKA and phosphorylation of the Ca²⁺ channel or an associated stimulatory type of regulatory protein. The cAMP-dependent PKA mediates most cAMP actions by phosphorylation. It is thought that in absence of cAMP, the two regulatory subunits associate with two catalytic units in the form of a tetramer in which the PKA catalytic activity is inhibited. Binding of the cAMP to the regulatory domain relieves this internal inhibitory effect, presumably by means of a conformational change causing the complex to dissociate into a dimer and two free active catalytic subunits. Phosphorylation of the catalytic domain is often required for the enzymatic activity of the PKA. It can be either constitutive, as in the autophosphorylation of Thr¹⁹⁷ in PKA, or provided by a regulatory kinase. The phosphorylations that occur on a loop in the vicinity of the catalytic site may play a critical role in the proper positioning of ATP and catalytic residues, as well as in the accessibility of the substrate (Knighton et al., 1991). The fine tuning of the cAMP cascade is provided by cAMP phosphodiesterases (PDEs) that break down cAMP and thus limit the degree of cAMP-dependent phosphorylation. Because PDE activity is controlled by the intracellular cGMP, both cAMP and cGMP are likely to determine the degree of cAMP-dependent phosphorylation of the cardiac Ca²⁺ in a competitive antagonist fashion. In ventricular myocytes from different species, I_Ca is probably deregulated by cGMP via a PDE-independent mechanism mediated by protein kinase G (PKG) and phosphorylation of possibly an inhibitory type of a regulatory protein associated with the Ca²⁺ channel (Sperelakis et al., 1994). Hence, the cGMP-PKG system stimulates a phosphatase that dephosphorylates the Ca²⁺ channel to provide a recycling mechanism for the channel. In addition to the slower indirect pathway exerted via cAMP-PKA, there apparently is a faster, more direct and efficient pathway for the stimulation of the Ca channels by -ARs that involves the direct modulation of the channel activity by the G_s protein (Knighton et al., 1991). The superiority in the efficiency of -AR over other systems in coupling to the regulation of cardiac I_Ca is thought to be embedded in the difference in the degree of cAMP accumulation near the Ca²⁺ channels due to colocalization of AC with the channels (Jurevicius and Fischmeister, 1996).

	IV. Cardiac Receptor Cross-Talk and -Adrenoceptor Signaling

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It is mandatory for an essential organ, such as the heart, to be endowed with the ability to fend off some of the potentially fatal consequences arising from possible failure or total loss of ₁-AR positive inotropism to sustain its very vital circulatory function in disease. The heart can achieve this by maintaining reserve signaling systems under its own humoral regulatory control or by the ability to adapt itself appropriately and rapidly to its altered circulatory demands. Moreover, several cardiac GPCR signaling components, including the G proteins, ACs, and PKs, exist in multiple isoforms and subtypes, of which different combinations determine the specificity of their actions. For example, different combinations of the G protein subunits often produce antagonistic signaling products when interacting with the various ACs. Thus, the AC V-VI group has G_s as their activator and G_i and Ca²⁺ act as inhibitors, whereas the complex appears to be uninvolved in this signaling process (Tesmer et al., 1997). Furthermore, G_s and G_o do not seem to inhibit certain ACs directly, apparently because this effect would be in opposition to those of their associated subunits (Tesmer et al., 1997). This diversity in the key components of GPCR signaling demonstrates the variability by which the different isoforms channel, refine, or divert signaling messages to meet the needs of the cardiac circulatory function according to demand.

One of the most intriguing phenomenon about -AR signaling is the manifestation that the cardiac contractile tissue harbors functionally viable ₂-AR in addition to the ₁-AR. Equall