1.2. Subtypes and Phylogenetic Expression of D₁-like receptors

The D₁ receptor was defined as a functional entity and characterized pharmacologically through the early work of Spano and colleagues (Spano et al., 1978), Kebabian and Calne (Kebabian and Calne, 1979), and others. As a molecular entity, however, it was not until the 1990s that the first member of the D₁-like subfamily of dopamine receptors was isolated almost concurrently by four different groups and named as the human D₁ receptor (Dearry et al., 1990; Sunahara et al., 1990; Grandy et al., 1990; Zhou et al., 1990) or the rodent D_1A receptor (Zhou et al., 1990; Monsma, Jr. et al., 1990). An additional D₁-like receptor was subsequently cloned in humans and termed D₅ (Grandy et al., 1991), while its counterpart in the rat was named D_1B (Tiberi et al., 1991). Afterwards, there was a spate of activity leading to the uncovering of multiple D₁-like transcripts in different organisms. Thus, current evidence indicates that D₁-like receptors are expressed in diverse species, including the fruit fly (Gotzes et al., 1994; Sugamori et al., 1995; Han et al., 1996; Reale et al., 1997; Schetz et al., 2003; Kehren and Baumann, 2005), cockroach (Orr et al., 1987; Evans et al., 1991; Evans and Green, 1991), locust (Homberg, 2002; Keating and Orchard, 2004), spider (Schmidt et al., 1981; Sauer et al., 1994), eel (Cardinaud et al., 1997), earthworm (Gardner and Cashin, 1975; Shpakov et al., 2008), flatworm (Venturini et al., 1989), goldfish (Mora-Ferrer et al., 1999), tree frog, (Agui et al., 1988; Schutte, 1991; Liu and Lasater, 1994; Behrens and Wagner, 1995) lizard (Clark et al., 2000), chicken (Agui et al., 1988; Demchyshyn et al., 1995; Schnabel et al., 1997; Sun and Reiner, 2000; Soares et al., 2000; Kubrusly et al., 2007), rodent (Grilli et al., 1988; Nisoli et al., 1988; Tiberi et al., 1991; Bryson et al., 1992; Mannoury la Cour et al., 2007), monkey (Besson et al., 1988; Sedvall et al., 1991; Choi et al., 1995; Bergson et al., 1995a; Bergson et al., 1995b) and humans (De Keyser et al., 1988a; De Keyser et al., 1988b; Dearry et al., 1990; Sunahara et al., 1990; Sidhu and Fishman, 1990; Grandy et al., 1990; Weinshank et al., 1991; Ferreira-de-Almeida et al., 1993). Among these species, multiple structurally divergent subtypes of D₁-like receptors have been detected, including at least 3 transcripts in Xenopus (D_1A, D_1B and D_1C), 4 in the chicken (D_1A, D_1B, D_1C and D_1D), 2 in the mouse or rat (D_1A and D_1B), 2 in the monkey, and 2 in humans (D₁ and D₅). Categorization of these proteins as D₁-like receptors is generally deduced from structural homologies among the transcripts, pharmacological interaction with selective dopamine D₁-like receptor ligands, and functional coupling to distinct stimulatory G-proteins and signaling cascades.

1.3. Peripheral and Brain Regional Distribution of D₁-like Receptors

At peripheral tissues of rodents or primates, D₁-like receptors are expressed in the heart (Ozono et al., 1997), in the walls of systemic arteries (Emilien et al., 1999), and in renal microvessels and proximal tubules (Girbes et al., 1992; Nash et al., 1993; Yao et al., 1998). Among these tissues, there are variations in the expression of D₁/D_1A and D₅/D_1B receptors. For instance, whereas the receptors in renal proximal tubules are of the D₁ type, those in systemic arteries and renal microvasculature appear to be of the D₅ subtype. How these differences in tissue distribution of the receptor subtypes may relate to the regulation of kidney function is not yet clearly understood.

Various mammalian brain regions show substantial but variable expression of D₁-like receptors. Based on immunodetection techniques, D₁/D_1A receptors are significantly expressed in the striatum, olfactory bulb, cerebral cortex, and to a lesser extent in the hippocampus and amygdala (Levey et al., 1993; Ariano and Sibley, 1994; Bergson et al., 1995b). On the other hand, the D₅/D_1B receptor in the rat or monkey shows a much more widespread expression across most brain regions, including the hippocampus, amygdala, frontal cortex, striatum, basal forebrain, thalamus, hypothalamus, cerebellum and brainstem (Bergson et al., 1995b; Ciliax et al., 2000). When both receptor subtypes are considered, the immunohistochemical data correlate generally well with the mRNA and receptor binding data. Curiously, though, there is a paucity of D₁-like receptor mRNA in the entopenducular nucleus, globus pallidus and substantia nigra pars reticulata of the rat, even though these regions demonstrate binding sites for D₁-like receptor ligands (Mengod et al., 1991). Thus, D_1A receptors, which have been specifically studied in these areas, may be synthesized elsewhere and then transported here, hence their confinement mostly to neuronal projections (Bergson et al., 1995b). Overall, the widespread distribution of D₅ receptors as opposed to the more confined presence of D₁ receptors in the basal ganglia suggests that these two members of the D₁-like subfamily could subserve nonidentical spectra of physiological functions.

1.4. Brain Cellular and Subcellular Localization of D₁-like Receptors

As with other GPCRs, D₁-like receptors are synthesized in the cell cytoplasm and are then subsequently transported to the plasma membrane of cell bodies and dendrites (Hersch et al., 1994; Liu and Lasater, 1994; Caille et al., 1996). Ultrastructural visualization studies on cortical tissues demonstrate the localization of D₁-like receptors in dendritic spines of medium spiny neurons as well as cortical pyramidal cells (Smiley et al., 1994; Hersch et al., 1995; Bergson et al., 1995b; Sesack et al., 2003). An extensive study by Bergson and colleagues (Bergson et al., 1995b) examined the regional, cellular and subcellular distribution of both D₁-like receptors in the primate brain. At the cellular level, medium spiny neurons of the neostriatum in primates showed immunoreactivity to both D₁ and D₅ antibodies, although the latter occurred to a lesser extent. Conversely, the large aspiny neurons which are typically cholinergic interneurons appeared to express only D₅ receptors. Differential localization of both receptor subtypes has also been clearly demonstrated in caudate and mesencephalic neuronal cell types. The largely separate localization of D₁ and D₅ receptors in medium sized spiny neuronal cells of the striatum has been suggested to arise either on account of variations in gene expression of each receptor subtype or as a result of differences in vesicular transport of the proteins (Bergson et al., 1995b). In cerebrocortical pyramidal cells, however, there is profuse reactivity to both subtypes of D₁-like receptors and experiments involving dual labeling with both subtypes show them to be commonly co-localized in cells within each region.

Whether expressed separately or in the same cells, D₁ and D₅ receptors are known to show distinct patterns of subcellular distribution (Bergson et al., 1995b). Generally, D₅ receptors are localized to neuronal perikarya and proximal dendrites, and occasionally in the neuropil of some tissues such as olfactory bulb, islands of Calleja, cerebral cortex, superior colliculus, and molecular layer of cerebellum (Ciliax et al., 2000). The D₁ receptors on the other hand are found mostly in axon terminals and dendrites (Bergson et al., 1995b). For individual cortical pyramidal neurons, D₁ receptors are expressed to a considerably higher degree in dendritic spines as opposed to the more prominent presence of D₅ receptors in dendritic shafts (Bergson et al., 1995b; Muly, III et al., 1998). Thus, D₁ and D₅ receptors are differentially expressed among brain tissues and demonstrate distinguishable patterns of subcellular distribution that probably reflect differences in signaling and physiological roles anticipated for each subtype.

2.2. Signaling via Adenylyl Cyclase

Adenylyl cyclase (AC) is a 12-transmembrane-spanning protein that catalyzes the conversion of adenosine triphosphate (ATP) to the intracellular second messenger cyclic-3′,5′-adenosine monophosphate (cyclic AMP). The activity of AC is regulated by Gs-type G proteins, notably Gs and Golf, and these G proteins are in turn regulated by ligand-induced receptor activation. Dopamine stimulates AC activity in various dopamine-innervated tissues in the brain or peripheral sites. Indeed, it was the discovery of dopamine-sensitive AC stimulation that led to the identification and subsequent definition of the “D1” (in contrast to the “D2”) dopamine receptor. As shown in figure 1, the stimulatory effect of the “D1” receptor on AC activity was found to be conveniently counterbalanced by an inhibitory effect mediated through activation of the “D2” receptor. The introduction of this schema provided a significant intellectual boost to the conceptualization and interpretation of experimental studies on dopamine neurobiology and pharmacology.

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Figure 1

Essentials of dopamine receptor coupling to adenylyl cyclase signaling. Stimulation of D1 dopamine receptors activates the G-protein Gs, or Golf, which activates adenylyl cyclase (AC) to convert ATP into the second messenger cyclic AMP (cAMP). The D2-like receptors couple through Gi to inhibit AC, resulting in decreased production of cAMP. The primary target of cAMP is protein kinase A (PKA). PKA phosphorylates and activates the transcription factors CREB (cAMP response element-binding protein), CREM (cAMP response element modulator) and ATF1 (activating transcription factor-1). PKA-mediated phosphorylation of DARPP32 converts the latter into a potent inhibitor of protein phosphatase-1 (PPtase1). Given that PPtase1 dephosphorylates, and therefore inhibits, ATF1, CREB, and CREM, the net effect of PPtase1 inhibition is to further enhance PKA-sensitive transcriptional activation. Conversely, PKA phosphorylates the L-type calcium channel, leading to increased cytosolic Ca2+, calcium-induced Ca2+ release from the endoplasmic reticulum, and activation of calmodulin (Calm) and protein phosphatase 2B/calcineurin A1 (PP2B/CalnA1). Calm and PP2B inhibit Thr34 activation of DARPP32, ultimately counteracting the transcriptional activating effect of PKA. This Ca2+- dependent effect provides opportunity for signaling crosstalk of the dopamine-PKA system with various calcium mobilizing receptors. Note that Ca2+ activation of PP2B also results in activation of casein kinase-1 (CK1), which in turn facilitates cyclin-dependent kinase-5 (CDK5) activation. As illustrated, CDK5 participates in DARPP32 regulation, thus underlining its role in diverse dopaminergic functions. Other abbreviations: SERCA (sarcoplasmic reticulum Ca2+- ATPase), IP3R (inositol trisphosphate receptor), CamK (calmodulin-dependent kinase).

3.1. Distinct Coupling Mechanisms for D₁ and D₅ Receptors

The first model is the rather conventional assumption that the D₁ and D₅ receptors employ a classical G-protein-transduced membrane machinery to couple to specific signaling pathways which are separately activated – implying that one pathway does not have to be active in order for the other to be activated. Either the same receptor molecule may select to couple to AC or PLC depending on its biological circumstance, or one receptor subtype couples to one and only one signaling cascade (AC or PLC) under physiologic conditions (but, like other G protein-coupled receptors, may become promiscuous under certain non-physiologic conditions such as when expressed in artificial cell lines or with an abundance of alternate G proteins). To facilitate discussion, we refer to these two variants as “distinct and selectable” (each subtype can choose AC or PLC as needed) and “distinct and separable” (one receptor subtype uses AC while the other uses PLC), respectively.

Notwithstanding their present pharmacological similarities, D₁ and D₅ receptors differ in several respects, as highlighted above. Depending on how physiologically substantive these differences turn out to be, they could provide the basis for a signaling model in which the two receptor subtypes distinctly couple through specific G proteins to regulate separate signaling cascades. In other words, it is possible that D₁ and D₅ receptors respectively couple to AC and PLC in physiological tissues. The “distinct and separable” signaling model, illustrated in Figure 3 for D₅, D₂, and D₁ receptor subtypes, is supported by a growing body of evidence.

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Figure 3

Schematic of dopamine-sensitive signaling via adenylyl cyclase (AC), phospholipase C (PLC), and phosphatidylinositol-3-kinase (PI3K) systems. This simplified view of the signaling pathways shows key downstream mediators and the various opportunities for interaction or integration among the pathways. Note the inhibitory effect of cyclic AMP-dependent protein kinase (PKA) on the activity of PLC, an action that provides a mechanism for crosstalk between the AC system and the PI/PLC systems. Besides hampering inositol trisphosphate (IP3)- mediated intracellular calcium mobilization (which would inhibit DARPP32 Thr34 phosphorylation via calmodulin/protein phosphatase 2B), it may also be possible that inhibition of PIP2 (phosphatidylinositol-4,5-bisphosphate) utilization via PLC could shunt the phospholipid to the PI3K pathway. Red arrows depict inhibition, while yellow arrows depict stimulation. Abbreviations: DA, dopamine; DRD1, Dopamine receptor D1 subtype; DRD2, Dopamine receptor D2 subtype; DRD5, Dopamine receptor D5 subtype; cAMP, cyclic AMP; PKA, protein kinase A; EPAC, Exchange protein activated by cyclic AMP; PPtase1, protein phosphatase 1, MEKs/ERKs, mitogen-activated kinases; ATF1/CREM/CREB/CDP, illustrative transcription-regulatory factors; CamK-II, calcium-calmodulin-dependent protein kinase type II; DG, diacylglycerol; IP3R, inositol trisphosphate receptor; Ca2+, calcium ions within and exiting the endoplasmic storage; PIP₃, phosphatidylinositol-3,4,5-trisphosphate; PTEN, phosphatase and tensin homolog; PDK-1, phosphoinositide-dependent kinase-1; Akt, protein kinase B; GSK3, glycogen synthase kinase-3; CcnD, cyclin D; mTOR, mammalian target of rapamycin; 4EBP1/eIF4E/p70S6K, downstream mediators to protein synthesis.

In physiologic tissues, the D₁ receptor has been consistently associated with AC signaling but not PLC signaling. Experimental manipulations that deplete or inactivate D₁ receptors result in the loss of dopamine-sensitive AC signaling but not dopamine-sensitive PLC signaling, suggesting that the latter response is not mediated via the D₁ receptor (Friedman et al., 1997; Undie et al., 2000). Further, dopamine and some D₁-like receptor agonists stimulate both the AC-coupled Gs and the PLC-coupled Gq proteins, but the receptor subtypes that mediate these responses appear to be separate. In a series of exquisite experiments by Friedman and colleagues (Wang et al., 1995; Friedman and Wang, 1996), it was demonstrated that dopamine and D₁-like agonists stimulated [³⁵S]GTPγS binding to the AC-coupled Gs and the PLC-coupled Gq proteins in rat striatal and frontal cortical tissues, but only Gαq was stimulated in the hippocampus or amygdala. When solubilized tissue membranes from the cortex or hippocampus were subjected to immunoprecipitation with anti-Gs or anti-Gq antisera followed by receptor binding analyses, SCH23390 binding sites were detected in both anti-Gs and anti-Gq-induced immunoprecipitates from the cortex, and in the anti-Gq precipitate from the hippocampus; however, only the anti-Gs-mediated immunoprecipitates also showed immunoreactivity for the D_1A receptor. This implies that the D₁-like receptor that is associated with the anti-Gq-induced immunoprecipitates is not the D_1A subtype. In these same experiments, it was noted that incubations with quinpirole resulted in the activation of Gi, but neither Gs nor Gq, hence arguing against the presence in this preparation of a D₁/D₂ oligomer that can stimulate Gq/PLC in response to either D₁-selective or D₂-selective agonists. These observations provide convincing evidence that the D_1A receptor mediates dopamine and D₁-like agonist stimulation of Gs/AC signaling but not the stimulation of dopamine-sensitive PLC signaling.

Current evidence for D₅ receptor coupling to AC signaling in native brain tissues is neither conclusive nor does it exclude the possibility of coupling to other signaling cascades. Indeed, following D_1A receptor knockout, there are extensive reductions (but not a complete loss) in D₁-like binding sites and loss of D₁-like agonist stimulation of AC (Mailman et al., 1986; Friedman et al., 1997; Montague et al., 2001). This may imply that the D₅ receptor contributes little to brain dopaminergic function or, more likely, that the D₅ receptor constitutes a smaller fraction of total SCH23390 binding sites in some brain regions such as the striatum, and that this site couples to a signaling cascade other than AC. With regard to relative receptor density, the fact that D₅ receptors are expressed mostly in neuronal perikarya and are localized intracellularly implies that a substantial portion of the receptor presence may not be detected in ordinary autoradiographic studies. Thus, the observation by Montague and colleagues (Montague et al., 2001) of near-total loss of [³H]SCH23390 autoradiography in D_1A knockout animals probably underestimates the true contribution of the D₅ subtype to brain D₁-like receptor populations and function. With regard to AC coupling, it is noted that following EEDQ-induced inactivation of SCH23390 binding sites in adult rats, whence there is as much as 75% loss of D₁ binding sites and complete loss of AC stimulation in response to D₁ agonists, there is no loss of D₁-like agonist stimulation of phosphoinositide hydrolysis (Rosengarten and Friedhoff, 1998; Undie et al., 2000). Hence, only a portion of D₁-like binding sites in the striatum may be required for mediating the full D₁-like agonist response on PLC in this tissue. Such would be consistent with the existence of functional spare receptors in D₁ systems (Battaglia et al., 1986; Meller et al., 1988; Watts et al., 1995). Alternatively, the observations may suggest the existence of different D₁-like receptor subtypes one of which is more susceptible to EEDQ and couples to AC while the other is less sensitive to EEDQ and couples to PLC. Were these two components of D₁-like receptors D₁ and D₅, respectively, then the ability of the D₅ receptor to promiscuously couple to multiple G proteins may explain its mediation of D₁-like agonist-stimulated cyclic AMP formation in some transfected cell lines (Sidhu et al., 1998b; Sidhu and Niznik, 2000).

The possibility that the D₅ receptor may be the subtype that mediates dopamine-sensitive phosphoinositide signaling was recently highlighted by observations in D₅ receptor knockout mice. Specifically, dopamine or D₁-like agonists failed to significantly induce IP accumulation or CDP-DG production in D₅-null mice compared to wild-type controls (Sahu et al., 2009), even though D₅ knockout animals have shown substantial D₁-like agonist stimulation of AC. Moreover, in experiments where intracellular imaging of agonist-induced phosphoinositide hydrolysis was assessed in organotypic striatal cultures, D₁-like agonist-induced CDP-DG accumulation was confined to the cell soma and proximal axons of the neurons (Undieh et al, Unpublished observations), which is consistent with the perikaryal subcellular distribution of the D₅ receptor but not the D₁ receptors that are typically localized to dendrites (Weiner et al., 1991; Yung et al., 1995). A D₅-PLC link has also been repeatedly implicated in studies of Gq activation or Ca2+ mobilization that permit separate molecular manipulations or observations of the D₁ and D₅ subtypes (Zheng et al., 2003; Baufreton et al., 2003; So et al., 2009).

The inference that the D₅ receptor may be the dopamine D₁-like receptor subtype that physiologically couples to PLC is consistent with the known characteristics of dopamine-induced phosphoinositide signaling and D₅ receptor expression among the brain regions. For instance, both receptor expression and agonist-induced signaling are relatively higher in the hippocampus than in the striatum, with intermediate effects in the prefrontal cortex (Undie and Friedman, 1990b). The D₅ receptor can couple to Gq-like G proteins in various cell lines or in renal brush border membranes (Sidhu and Niznik, 2000), the receptor directly modulates calcium currents and burst firing in the subthalamic nucleus (Baufreton et al., 2003), and it frequently exists in extrasynaptic microdomains associated with neuronal inositol-1,4,5-trisphosphate-sensitive calcium stores (Paspalas and Goldman-Rakic, 2004). The D₅ inference is also supported by previous experiments involving the expression of a D₁-like receptor encoded by striatal mRNA in Xenopus oocytes. Mahan and coworkers (Mahan et al., 1990) found that injection of rat striatal mRNA into Xenopus oocytes led to the expression of a D₁-like receptor coupled to inositol phosphate production and Ca²⁺ mobilization. However, expression of the cloned rat D₁ receptor in the oocytes led to the production of cyclic AMP, but not Ca²⁺ mobilization, suggesting that the D₁ receptor was not linked to the phosphoinositide response (Monsma, Jr. et al., 1990). Moreover, using size fractionation techniques, it was shown that mRNA encoding the striatal phosphoinositide-linked D₁-like receptor was between 2.5-3.0 kb in size, thus distinguishing it from the 4.1 kb mRNA fragment that encodes the rat D₁ receptor. Interestingly, the rat D₅ receptor is encoded by an mRNA that is ~3 kb in size (Tiberi et al., 1991), in close agreement with the size of the mRNA encoding the phosphoinositide-linked D₁-like receptor identified using the oocyte expression system. Hence, the Ca²⁺ response observed in those early oocyte expression experiments probably involved the D₅ receptor subtype.

A separate coupling of D₁ and D₅ receptors to distinct signaling cascades is more in tune with numerous distinct functional observations such as: (i) the opposite effects of these receptors on LTP v. LTD (Centonze et al., 2003a), (ii) differential intrastriatal distribution of the receptors among chemically and functionally different neuronal phenotypes (Le Moine et al., 1991; Kawaguchi et al., 1995; Rivera et al., 2002; Centonze et al., 2003b), (iii) differential interactions with other receptors and neuromodulators (White et al., 1999; Liu et al., 2000), (iv) effects of antisense oligonucleotide-induced downregulation on signaling responses in adult animals (Undie, 1998), (v) regulatory actions on hypothalamic function (Apostolakis et al., 1996a), (vi) modulation of lordosis following specific antisense oligonucleotide treatments and local application of agonists in the brain (Apostolakis et al., 1996b), (vi) effects on motor control (Sibley, 1999; O’Sullivan et al., 2004); and (vii) significantly different phenotypes resulting from genomic inactivation of the receptors (Waddington et al., 1995; Nicola et al., 1996; Waddington et al., 2001; Holmes et al., 2001).

The foregoing inferences present a new opportunity to interpret or reinterpret the role of the D₅ receptor as the D₁-like receptor subtype that mediates a subset of dopamine’s effects. Such effects that were previously held suspect might include the regulation of peripheral blood pressure (Hollon et al., 2002), enhanced acetylcholine release in the hippocampus (Hersi et al., 2000), stimulation of pituitary prolactin secretion (Saller and Salama, 1986; Fabbrini et al., 1988; Schoors et al., 1991), and modulation of mucosal vulnerability to psychosomatic ulcerogenic insults (Hunyady et al., 2001). At the behavior level, congenic D5 receptor mutant mice show marked reductions in grooming, a characteristic D₁-like dopaminergic response that is nevertheless not cyclase-mediated (O’Sullivan et al., 2005). Moreover, orofacial movement topographies inducible in naïve animals by SKF83959 (which does not stimulate AC), are severely disrupted in congenic D₅ mutant mice (Tomiyama et al., 2006). Nevertheless, such mice continue to demonstrate grooming and episodic seizure activity in response to the AC-effective D₁-like agonist, SKF83822. Hence, the loss of D₅ signaling correlates more to behavioral mediation via PLC than via AC, thus providing further support to the notion that the D₅ receptor regulates a defined subset of physiological dopaminergic responses, as was previously thought for the dopamine-linked phosphoinositide signaling response (Deveney and Waddington, 1995; Adachi et al., 1999; Clifford et al., 1999; Undie et al., 2000; Makihara et al., 2007).

In sum, then, the evidence favours the distinct and separable coupling of D₁/D_1A receptors to AC signaling and of the D₅/D_1B receptor subtype to PLC signaling in native brain (and possibly renal) tissues.

3.2. Activity-Dependent Selectivity of Signaling Pathways

This model seeks to interpret observations that have revealed significantly different responsiveness between the dopamine-sensitive AC and PLC cascades. In general, a primary messenger may activate multiple and distinct transmembrane signaling pathways to achieve diverse functional outcomes within a given tissue, provided that the signaling pathways are sufficiently differentiable in sensitivity and responsivity. In this context, sensitivity is the inverse of the ligand concentration (or stimulation frequency) necessary to elicit a biologically or statistically significant (or 50% of maximal) response; while responsivity refers to the efficiency with which receptor activation is translated into a physiological response – mathematically inferred from the maxima of the concentration (or stimulation frequency)-response curve. The concentration of ligand at which either system attains maximal response may differ between the systems; however, the magnitudes of the response maxima for the two signaling systems are irrelevant to the operation of the model (seeing, for example, the unlikelihood that any biological system would need to routinely function at maximum levels). Thus, sensitivity and responsivity could be as much a property of the signaling system as potency and efficacy are properties of a pharmacological ligand. Physiologically, this sort of model would be applicable where one signaling system is deployed to regulate function at tonic to moderate levels of neuronal activity and the second system is mobilized to supplement or supplant the first system during occasions of high neuronal stimulation.

For the dopamine system, the activity-dependent selectivity model implies that D₁-like receptor signaling through AC and/or PLC pathways is determined by the intensity and duration of synaptic dopamine activity. Thus, at tonic to moderate levels of dopamine neuron stimulation (or agonist concentration), one pathway is active, whereas at higher levels or longer durations of stimulation the second pathway is mobilized to supplement or supplant the base system. While it is not necessary to the operation of this model that each signaling pathway be activated through a different subtype of D₁-like receptors, such is not precluded either. However, the receptor subtypes, conformations, or states that couple to the different pathways must be differentiable in terms of sensitivity and/or responsivity. Indeed, from a design perspective, using separate receptor subtypes might provide for more optimal regulation of the system, for example, by deploying differential density of expression, cellular and subcellular distribution, or sensitivity to modulation by co-transmitters. Several experimental observations lend credence to the possible operation of this model within the dopamine system.

Pharmacological studies of AC and PLC signaling reveal concentration-dependent effects of dopamine and selective D₁-like agonists at each signaling response (Undie et al., 1994). Agonist potencies between the two pathways, however, can differ by as much as an order of magnitude (Undie et al., 1994). Generally, the AC system shows the greater sensitivity. Some of the sensitivity difference may be accounted for by the experimental use of whole cells or tissue slices for PLC assays versus membrane preparations for AC assays, and by the availability of good “traps” (i.e., phosphodiesterase inhibitors) for cyclic AMP versus poorly effective “dams” (LiCl) for inositol phosphate or diacylglycerol analytes. Nevertheless, when both the AC and PLC assays were conducted in brain slice preparations, the AC response still saturated at lower dopamine concentrations than the PLC response, although the PLC response showed the greater fold increase above control (Undie and Friedman, 1994; Wang et al., 1995; Panchalingam and Undie, 2005). Additionally, AC stimulation appears to attain maximal response within minutes, whereas PLC stimulation, measured as accumulation of either inositol phosphates or CDP-diacylglycerol, continues to show increases up to an hour or longer (Undie and Friedman, 1990b; Undie and Friedman, 1994; Undie, 1999). Thus, not only must sensitivity and responsivity (or potency and efficacy) be considered, we must also integrate a temporal factor in the mix of parameters that may functionally differentiate the two D₁-like receptors or dopamine-sensitive signaling pathways.

Temporal studies of agonist-stimulated GTP binding, an assay that indicates effective receptor stimulation provide support for the activation-driven selectivity model. D₁-like agonist stimulation of Gs, the AC-coupled G protein, occurs relatively fast and reaches peak levels of activation within 15 min (Wang et al., 1995). Conversely, D₁-like agonist stimulation of the PLC-coupled Gq occurs with a lag time of 10-15 min, and attains peak responses only after 60 min (Wang et al., 1995; Panchalingam and Undie, 2005). Nevertheless, the eventual maximal percentage stimulation of Gq is several fold greater than the maximal percentage stimulation of Gs (Panchalingam and Undie, 2001; Panchalingam and Undie, 2005). Table 1 shows summary data indicating that assaying Gq protein activation at longer time points can “increase” the measurable response from 0-20% above control seen by 15 minutes to 400-700% above control as seen after 120 min. The integration of comparable agonist concentrations over a longer duration of activation is apparently more favorable to Gq/PLC coupling in contrast with the coupling to Gs/AC. This brings to the forefront a question about the role of time – duration – in functional responsivity. Is time important only to allow for adequate diffusion of drug to the site of action, or may signaling events cumulate with time so as to attain a threshold or extend a response? For instance, Gq-mediated PLC stimulation usually consists of a fast and transient phase followed by a slower but prolonged phase. While this is easily explicable on the basis of diffusion-related impediments in the case of whole cells or organ systems, the fact that these temporal patterns can occur even in cell-free preparations implies that they may be a property of the signaling apparatus rather than the result of physicochemical impediments to drug diffusion (Thorne and Nicholson, 2006; Thorne et al., 2008). Such a phenomenon of cumulative signaling should warrant further exploration. But, for the present discourse, it is intriguing that dopamine could achieve a level of functional sequencing by employing two receptor subtypes that signal through a sensitive, fast-onset, short-duration cascade such as the AC system and a comparably less sensitive, slower-onset and longer-duration system such as the PLC system.

Another line of support comes from a 1995 study showing that elevation of intracellular cyclic AMP inhibits subsequent D₁-like agonist stimulation of phosphoinositide hydrolysis (Undie and Friedman, 1994). To obtain the same levels of inositol phosphate stimulation, agonist concentrations had to be raised. Cyclic AMP-induced inhibition of PLC signaling is evident when cellular cyclic AMP levels are increased by incubations with cell-permeable cyclic AMP analogs, by in situ activation of AC with forskolin, or by treatment with Gs-effective dopaminergic agonists that stimulate cyclic AMP (Undie and Friedman, 1994). As such, blockade of cyclic AMP action with RpcAMPS can release the system from cyclic AMP-mediated inhibition, allowing Gq-effective D₁-like agonists to induce significantly greater activation of PLC. Co-stimulation of phosphoinositide hydrolysis, however, does not appear to modulate subsequent cyclic AMP formation. Taken together, these findings suggest that the cyclase system may be attuned to function at tonic to moderate levels of dopaminergic activity, whereas colocalized Gq-mediated activity may not be evident until moderate to high levels of dopaminergic stimulation are attained. Such higher levels of synaptic dopamine may be achieved endogenously during high phasic neuronal stimulation or following administration of agents like psychostimulants that inhibit dopamine clearance resulting in markedly elevated synaptic levels of the transmitter.

A series of neurochemical and functional studies from different laboratories indicate that the response profiles obtained by the activation of D₁-like receptors can vary qualitatively depending on the amount of agonist administered (Cai and Arnsten, 1997; Zahrt et al., 1997; Arnsten, 1997; Granon et al., 2000; Lidow et al., 2003). Rather than induce quantitatively commensurate responses until a peak is attained, varying the agonist concentration past a critical point could rather elicit divergent or opposite effects on a given functional endpoint. Calabrese and colleagues have applied the term hormesis to the phenomenon in which a substance ordinarily evoking stimulatory responses at low doses is found to generate inhibitory responses at high doses or vice versa (Calabrese and Baldwin, 2002). Hormesis exists in two general forms of dose response relationships: the monotonic dose response which involves responses that generally proceed from zero or above and move on unidirectionally, and the bitonic dose response which presents a bi-directional or biphasic form where the dose response curve assumes either a U, inverted U, or J-shape, perhaps depending on the included dose segments of measurement (Calabrese, 2002). Stimulation of D₁-like receptors in monkeys and rats executing working memory tasks associated with the prefrontal cortex has been shown to produce an inverted U-shaped dose-response curve (Lidow et al., 2003). In this instance, either suboptimal stimulation (Sawaguchi and Goldman-Rakic, 1991; Seamans et al., 1998; Kozlov et al., 2001) or over-activation (Zahrt et al., 1997; Arnsten and Goldman-Rakic, 1998) would tend to interfere with task execution. Other studies in monkeys have shown similar inverted U responses to D₁-like receptor activation by delay-related activity of prefrontal cortical neurons which are strengthened at low but attenuated at high doses (Sawaguchi and Goldman-Rakic, 1994; Williams and Goldman-Rakic, 1995). A notable observation about hormesis in D₁-like receptor function is that it occurs with D₁-like receptor stimulation under relatively normal physiology. Thus, an ability to mobilize an alternate signaling cascade that supplants the base system under excessive levels of activation could serve as a self-managed circuit-breaker of sorts. Indeed, it has been speculated that this phenomenon may underlie some of the ordinary differences observed in higher cognitive abilities (Vijayraghavan et al., 2007). While the neurophysiological or molecular explanations for these phenomena are yet to emerge, constructs such as the present activity-dependent selectivity hypothesis could help to systematize future investigations to help link molecular mechanisms with functional outcomes.

3.3. Dynamic Compartmentalization of Signaling Components

The existence of multiple membrane folds and compartments within the cell creates extended surfaces or pockets for inserting various molecules whose juxtaposition or segregation might be crucial for normal cell function. Although most GPCRs including D₁-like receptors are generally seen as being localized on the plasma membrane, at basal cellular conditions the majority of these receptors occur mainly in intracellular compartments, as visualized in kidney and heart cells (O’Connell et al., 1995; Ozono et al., 1996; Brismar et al., 2002; Kruse et al., 2003), and in native brain tissues (Bloch et al., 1999). This raises the possibility that, in a cell expressing multiple receptor subtypes, subpopulations of the same receptor subtype or members of separate receptor subtypes could be localized to different subcellular compartments. From here the receptors may traffic to the cell membrane as needed, or while within the intracellular compartment the receptors could participate in local intracellular signaling events that could be equally significant to normal cell function (Bloch et al., 1999; Sadowski et al., 2008). In rat cerebrocortical tissues, D₁ and D₅ receptors appear to differentially distribute to distinct subcellular compartments, apparently facilitated and abated by the presence of lipid rafts and reductive thiol functions (Voulalas and Undieh, Unpublished observations). Interestingly, dopamine or a D₁-like receptor agonist could induce inter-compartmental redistribution of the D₁ but not the D₅ (or even the D₂) receptor subtype. This may imply that there are functional consequences emanating from the subcellular localization of dopamine receptor subtypes.

Lipid rafts appear to be critical for maintaining the intracompartmental distribution or segregation of receptors and other signaling elements and membrane proteins. The primary constituents of these membranous microdomains are structural lipids, notably sphingolipids, cholesterol, and glycolipids; in addition there are specific proteins whose major distinguishing features are their insolubility in nonionic detergents at 4 °C as well as their light buoyancy on sucrose gradients (Little and Teyler, 1998; Ming et al., 2006). Lipid rafts are thought to provide an enabling environment for the formation of functional complexes among various molecular components of signal transduction cascades (Lisanti et al., 1994; Simons and Ikonen, 1997; Shaul and Anderson, 1998; Ostrom et al., 2000; Galbiati et al., 2001; Anderson and Jacobson, 2002). It has been possible to isolate from raft-like structures various signaling molecules that are known to be involved in D₁-like receptor cascades, including heterotrimeric G-proteins, GTPases, GRK, AC, PLC, PKC and PKA (Sargiacomo et al., 1993; Smart et al., 1995; Song et al., 1996; Carman and Benovic, 1998; Bathori et al., 1999; Razani et al., 1999; Carman et al., 1999; Ostrom et al., 2000). While these mediators are not necessarily restricted to dopaminergic systems, their presence in these cellular substructures further attests to the significance of membrane molecular segregation or packaging in cellular signaling function.

Among the numerous varieties of lipid rafts that have been identified, the best known are the caveolae, which are polymerization products of caveolin and cholesterol occurring primarily on invaginations of cellular surfaces (Lisanti et al., 1994; Shaul and Anderson, 1998; Ostrom et al., 2000; Galbiati et al., 2001; Anderson and Jacobson, 2002). Caveolae facilitate the creation of subcellular compartments for various signaling constituents such as adaptors, scaffolds and enzymes; such compartmentalization enhances the competency and efficiency of receptor coupling to multiple effector systems (Lisanti et al., 1994). In this regard, Trivedi and colleagues (Trivedi et al., 2004) showed that functionally competent D_1A receptors with the conserved ability to couple with G-proteins as well as stimulate cyclic AMP accumulation and inhibit Na/K-ATPase in rat proximal tubules are conscripted to caveolar plasma membranes rich in Na/K-ATPase by the agonist action of dopamine. The dopamine agonist action appears to be mediated through D₁-like receptor-cyclic AMP signaling pathways. In other experiments, Yu and coworkers (Yu et al., 2004) uncovered a link between caveolin-2β and human D₁-like receptors whereby stimulation with fenoldopam, a D₁-like receptor agonist, led to an increase in the amounts of caveolin-2β that were associated with the receptors. Agonist-induced D₁ receptor-mediated AC activation was observed at much higher degrees in lipid rafts as opposed to plasma membranes not associated with lipid rafts. Moreover, following antisense-mediated knockdown of caveolin-2 expression, the agonist action of fenoldopam on cyclic AMP production was significantly reduced. Based on these studies, it appears that the primary location of human D₁ receptors, at least in these cells where they were exogenously expressed, is in lipid rafts and that heterologous as well as endogenous expression of these receptors might be closely linked to and modulated by caveolin-2. In contrast, the D₅ receptors are predominantly intracellularly localized and their behaviour in response to agonist stimulation remains to be fully elucidated.

Besides their differential steady-state localization, D₁-like receptors traffic between the cell membrane and other cellular compartments, and these movements are relevant to ultimate receptor function. D₁-like receptors as other GPCRs, are synthesized cytoplasmically and then are transported to the plasma membrane of cell bodies and dendrites (Levey et al., 1993; Hersch et al., 1994; Liu and Lasater, 1994; Caille et al., 1996). Within minutes following acute activation by dopamine, D₁-like receptor localization is further modified by translocation of the receptors from neuronal surfaces to cytoplasmic endosomal compartments with consequent reduction in the density of membrane receptors (Mantyh et al., 1995a; Mantyh et al., 1995b; Sternini et al., 1996; Dumartin et al., 1998; Bernard et al., 1998; Bloch et al., 1999). Subsequently, the internalized receptors are degraded or are resensitized by being recycled back into the plasma membrane. The complex process of intracellular trafficking contributes to the regulation of the abundance of D₁-like receptors at the cell surface where typically receptor-ligand interactions occur. Of additional interest is the possibility that trafficking of D₁ and D₅ receptors, or of AC-coupled and PLC-coupled sites, may differ, with attendant functional implications. For example, Kruse and colleagues (Kruse et al., 2003) showed that dopamine-induced D₁-like receptor translocation from intracellular compartments to the plasma membrane is associated with altered activation of protein kinase C. This finding is consistent with our own recent observation that nocodazole and other microtubule inhibitors block dopamine or D₁ agonist stimulation of PLC signaling in brain tissue probably by preventing cytoskeletal transport of mediators to the cell membrane (Undieh et al, Unpublished observations). While a clear understanding of these observations must await additional mechanistic studies, the observations are nevertheless consistent with the view that diverse signaling mediators are segregated among subcellular compartments, and that the regulated movement of the molecules among the compartments may be a natural and usual concomitant of receptor activation.

For receptors that could interact with multiple signaling partners, the subcellular localization of the receptor could become an important determinant of the receptor’s signaling response in certain cells or under particular conditions. As an example, following expression in intracellular compartments of COS-7 cells, the D₁ receptor constitutively heteromerizes with the NR1 subunit of the NMDA receptor, whereas the D₅ receptor does not undergo any such interaction (Fiorentini et al., 2003). This difference, which is attributed to differences in the C-terminal structure of the two receptors, enables the D₁ receptor to be primarily targeted to the plasma membrane, to co-exist with the NMDA receptor in the postsynaptic density, and to resist agonist-induced cytoplasmic sequestration (Fiorentini et al., 2003). On the other hand, N-glycosylation is critical for functional cell surface expression of the D₅ receptor in HEK-293 cells, whereas inhibition of N-glycosylation has no effect on trafficking of the D₁ receptor to the plasma membrane (Karpa et al., 1999). As earlier highlighted, these kinds of differences between the D₁ and D₅ receptors are probably not accidental, but rather appear to be consistent with (or even deterministic of) the differing functional roles of the receptors. Future studies to further clarify these potential distinctions should incorporate experimental designs that deliberately compare or contrast D₁ versus D₅ receptor subtypes as well as AC-mediated versus PLC-related mediators and end points. For now, it remains speculative that signaling differences could result from relative differences in receptor subtype expression in a given tissue or cell, trafficking to the cell membrane, subdistribution into lipid rafts or other membrane compartments, localization within or near synaptic clefts, functional selectivity for a given test ligand (Ryman-Rasmussen et al., 2007), association with any receptor activity-modifying proteins, propensity for cross-phosphorylation, readiness to undergo intracellular sequestration, and susceptibility to endosomal degradation or functional recycling to active sites on the cell membrane.

3.4. Receptor Oligomerization for Recruitment of Alternate Signaling Cascades

Structural and biochemical evidence points to the innate capacity of diverse GPCRs to endogenously form molecular complexes consisting of two subunits (dimers) or multiple subunits (oligomers). The subunits may structurally represent the same receptor entity (hence, homomers) or the units may come from two distinct receptors belonging to the same or different families (heteromers) (Gouldson et al., 1998; Hebert et al., 1998; Bouvier, 2001). Dopamine D₁ receptors can form homodimers, heterodimers, and possibly oligomers as well (Seeman et al., 1992; George et al., 1998; Lee et al., 2000; Liu et al., 2000; O’Dowd et al., 2005; Kong et al., 2006). Interestingly, if our observation of subcellular segregation of D₁ and D₅ receptors in neocortical neurons can be generalized to other tissues, then it is unlikely that these two species would be able to come close enough to form a D₁-like heterodimer under normal circumstances in such tissues. Nevertheless, D₁-like receptors, especially the D₁ receptor, enjoy a rich repertoire of heteromerization with D₂-like dopaminergic, A₁ adenosine, and other receptor species (Franco et al., 2000; Lee et al., 2004; So et al., 2005; Rashid et al., 2007a; Juhasz et al., 2008).

While homodimerization of D₁-like receptors could enhance functional cooperativity particularly involving receptor stimulation and signal transduction amplification (Kong et al., 2006), heteromerization is thought to be motivated by a need to generate functional complexes with agonist affinity and signaling mechanisms separate from those of the original individual receptor subunits (George et al., 2002; Kong et al., 2006). Nevertheless, signal amplification may not be the exclusive rationale for homomerization, hence both homomers and heteromers may modulate receptor trafficking (Nelson et al., 2001; Uberti et al., 2003; Hague et al., 2004; Kong et al., 2006), and intracellular localization (O’Dowd et al., 2005), as well as serve a signal-enhancing function by increasing the number of receptor units that can be available for activation by a single ligand or agonist molecule.

The functional consequences of D₁ receptor heteromerization with D₂ receptors has been explored by George and colleagues (Lee et al., 2004; So et al., 2005; Rashid et al., 2007b). Notably, D₁/D₂ oligomers are detectable under unstimulated conditions, and demonstrate patterns of cell surface localization, internalization, and transactivation that are distinct from the properties of homooligomeric D₁ or D₂ receptors (So et al., 2005). Functionally, the D₁/D₂ heteromeric complex responds additively to selective D₁ and D₂ agonists, and elicits a novel Gq/phospholipase C/Ca²⁺ signal that is not seen with either receptor separately expressed and stimulated by its cognate agonist (Lee et al., 2004; Rashid et al., 2007b). It has recently been demonstrated that the D₅ receptor also can oligomerize with the D₂ receptor (So et al., 2009). However, in contrast with D₁/D₂ oligomerization where agonist-induced calcium mobilization is facilitated, D₅/D₂ heteromerization results in inhibition of D₅-mediated calcium mobilization. This is consistent with our recent demonstration that the D₅ receptor natively couples to Gq/PLC signaling (Sahu et al., 2009) and that a knockout of the D₂ receptor does not alter D₁-like agonist stimulation of phosphoinositide hydrolysis (Undieh et al; Unpublished observations). Thus, it is demonstrated once again that D₁ and D₅ receptors may show close similarities with regard to ligand selectivity, but the structural differences at the C terminal tails of these two D₁-like receptors exert significant influences on the molecular interactions and signaling outputs of the receptors.

While several models have been proposed to explain the general intermolecular interactions that lead to receptor oligomerization (Bouvier, 2001), it is noteworthy that for the D₅ receptor a C-terminal segment appears to be the portion that engages in interactions with the GABA_A receptor. Hence, the full length receptor may not be required in order for oligomeric interactions to occur. Also, George and colleagues (George et al., 1998) demonstrate that a peptide homologous only to the t6 transmembrane domain of the D_1X receptor inhibited D₁ receptor function. Hence, one wonders if similar regulation might exist for the D₅ receptor, and if this might hint at a possible biological significance for the existence of D₅ receptor pseudogenes. Two of these pseudogenes which code for non-functional abbreviated forms of the D₅ receptor have been detected and characterized to be about 98 % identical to each other and 95 % identical to the D₁ receptor (Weinshank et al., 1991; Grandy et al., 1991; Grandy et al., 1992). Might physiological or pathological expression of the receptor pseudogenes serve a role in modulating authentic D₅ receptor expression, intracellular compartmentalization, trafficking to the plasma membrane, affinity for dopamine ligands, or coupling to specific signal transduction cascades?

The thought then is that even if the known D₁-like receptors individually coupled to similar signaling cascades, their divergent oligomerization with distinct populations of heterologous receptors could produce sufficiently differentiated signaling outcomes to justify their co-expression (and hence simultaneous stimulation by dopamine) in various tissues.