To our knowledge, the present results provide the first analysis of the relationship between D1 receptor trafficking and signaling in neurons, and on a time scale approaching that of physiological dopaminergic neurotransmission. Our results demonstrate that D1 receptors enter the endocytic pathway within ~1 min after activation by either DA or synthetic agonist and that receptor-mediated accumulation of cellular cAMP occurs with overlapping kinetics. They also establish a causal relationship whereby D1 receptor endocytosis augments acute dopaminergic signaling. We demonstrate that recycling is not required for this response and provide evidence that the endocytosis-dependent signal is generated from an early endosomal membrane, thus distinguishing the present results from endocytosis-dependent resensitization observed for several other GPCRs. Further, our results show that the endocytosis-dependent component of the D1 receptor-mediated signal is functionally relevant as it is required to increase AP firing in a native brain slice preparation.
Previous studies of D1
receptor-mediated signaling effects, measured over longer time intervals (> 30 minutes), have suggested that endocytosis either inhibits (Jackson et al., 2002
; Zhang et al., 2007
) or has no effect on dopaminergic signaling (Gardner et al., 2001
). Further, endocytosis has been generally shown to attenuate cellular responsiveness, such as for AMPA receptor endocytosis and LTD (Malenka, 2003
), or restore cellular responsiveness, such as for resensitization of β-adrenoreceptors after prolonged stimulation or stimulation and a refractory period (Pippig et al., 1995
). We are not aware of previous evidence that endocytosis augments the acute cAMP response mediated by any signaling receptor. Our study focused on cells in which D1
receptors are primarily thought to endocytose via clathrin-coated pits. There is evidence that caveolae mediate a slower component of D1
receptor endocytosis in other cells, but this is not thought to affect the acute cAMP signal (Kong et al., 2007
). As such, we believe that the presently identified role of endocytosis in supporting acute D1
receptor-mediated signaling is unique.
What is the mechanism by which rapid endocytosis contributes to dopaminergic signaling? We initially favored the hypothesis that this augmentation might occur by rapid cycling of receptors back to the plasma membrane. This was motivated by analogy with the resensitization paradigm established for several other GPCRs. As presently understood, however, the resensitization paradigm explains recovery of signal responsiveness after prolonged or repeated activation, and does not affect the acute signaling response (Lefkowitz, 1998
; Pippig et al., 1995
). Nevertheless, given the rapid kinetics with which D1
receptors were found to traverse the recycling pathway, we considered the hypothesis that D1
receptors might recycle so rapidly that their resensitization might have been missed by the previous paradigm. We rejected this hypothesis because genetic (EHD3 knockdown) and chemical (bafilomycin A1
) inhibition of the recycling pathway did not affect D1
receptor-mediated cAMP accumulation, in contrast to the pronounced inhibition produced by various endocytic inhibitors.
Our results thus support the alternative hypothesis that endocytosis augments acute dopaminergic signaling by facilitating direct D1
receptor-mediated signaling from a membrane domain in the early endocytic pathway (Figure 9E). This is supported by immunocytochemical localization data showing close proximity between D1
receptors and downstream transduction machinery upon initial entry to the endocytic pathway. While there is no doubt that the plasma membrane is a major site of GPCR signaling, there is emerging evidence that signaling can also occur from the endocytic pathway, and there is presently no compelling reason to rule out endosomal signaling via trimeric G proteins (Calebiro et al., 2010
; Sorkin and von Zastrow, 2009
). Trimeric G proteins have previously been detected on endomembane compartments in mammalian cells and tissues (Marrari et al., 2007
). Further, G protein α-subunits can mediate functionally significant signaling from endosomes in yeast (Slessareva and Dohlman, 2006
). Recent evidence suggests that two other mammalian GPCRs (the TSH and PTH receptors) signal via G protein-linked activation of AC directly from the endosome membrane (Calebiro et al., 2009
; Ferrandon et al., 2009
). However, the endocytosis-dependent component of signaling described for these GPCRs is specific to a component of the cAMP response measured tens of minutes after initial receptor activation. Our studies establish a novel role of the endocytic machinery in augmenting rapid D1
receptor-mediated signaling, and show that D1
receptors achieve close proximity to essential downstream signaling components upon or shortly after endocytosis. It remains to be determined whether endomembrane signaling of D1
receptors is more effective than signaling from the plasma membrane, or if endocytosis restores signaling activity after termination at the plasma membrane.
The present discovery has intriguing implications for neuroscience. A variety of complex functions including learning and memory, locomotion and goal-directed behaviors such as food or drug seeking require precise regulation of dopaminergic signaling via D1
receptors (Kelley, 2004
; Sibley, 1999
). Further, recent studies in awake, behaving animals have shown transient spikes in DA concentrations that last on the order of seconds (Heien et al., 2005
; Roitman et al., 2008
; Tsai et al., 2009
). Our data indicate that robust endocytosis of D1
receptors can occur, and is capable supporting cellular cAMP signaling on a time scale that approaches this physiology.
In vivo measurements have shown that extracellular DA transients can vary in peak intensities from nano- to micromolar concentration. We have demonstrated that the D1
receptor undergoes rapid endocytosis in the upper range these concentrations (see
). Interestingly, the peak concentration measured in each of these studies varied substantially depending on the experimental paradigm that elicited DA transients. Rewarding taste stimuli evoked DA transients in the nucleus accumbens with peak concentrations near 50nM (Roitman et al., 2008
), whereas electrical stimulation of dopaminergic VTA neurons could elicit DA transients in the nucleus accumbens with peak amplitudes greater than 0.5 μM (Heien et al., 2005
). In vivo microdialysis measurements of DA in the striatum of non-human primates showed dopamine concentrations greater than 1uM after self-administration of cocaine (Bradberry et al., 2000
). Our data predict that low extracellular DA concentrations within the brain would stimulate little D1
receptor endocytosis, while higher extracellular DA concentrations would elicit robust D1
receptor endocytosis and promote acute ongoing dopaminergic signaling. Thus, endocytosis of D1
receptors may reflect a novel mechanism by which signal strength, signal duration and perhaps even the salience of a given stimulus could be effectively encoded at the cellular level.
It is important to note that although our studies examined D1 receptor mediated signaling with substantially improved temporal resolution relative to conventional biochemical approaches, we are not yet capable of resolving effects with the kinetics of physiological dopamine release thought to occur within the healthy brain. Thus our findings may be more applicable to drug-induced or pathological states. Future studies, delivering dopamine in a more transient manner and further improving the temporal resolution of analysis, will be important to investigate this issue.
In conclusion, the present study examined rapid D1 receptor-mediated signaling and endocytic trafficking and identified a role of the endocytic machinery in supporting a component of acute dopaminergic signaling. We believe that these findings establish a previously unanticipated relationship between the endocytic machinery and acute cAMP signaling, and do so in neurons that naturally respond to DA. We propose that endocytosis-supported signaling by D1 receptors likely represents a fundamental principle by which the nervous system shapes and maintains dopaminergic responsiveness at the level of the individual neuron.