The PDZ Binding Motif of the β1 Adrenergic Receptor Modulates Receptor Trafficking and Signaling in Cardiac Myocytes

Abstract

␤ 1 and ␤ 2 adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse cardiac myocytes through distinct signaling pathways. It has been shown that stimulation of ␤ 1 ARs leads to a protein kinase A-dependent increase in contraction rate. In contrast, stimulation of ␤ 2 ARs has a biphasic effect on contraction rate, with an initial protein kinase A-independent increase followed by a sustained decrease that is blocked by pertussis toxin. The ␤ 2 AR undergoes agonist-induced endocytosis in cardiac myocytes while the ␤ 1 AR remains on the cell surface. It has been shown that a PDZ domain binding motif at the carboxyl terminus of ␤ 1 AR interacts with the postsynaptic density protein PSD-95 when both are expressed in HEK293 cells. We found that mutation of this PDZ binding motif in the ␤ 1 AR (␤ 1 AR-PDZ) enabled agonist-induced internalization in cardiac myocytes. Moreover, stimulation of ␤ 1 AR-PDZ had a biphasic effect on the myocyte contraction rate similar to that observed following stimulation of the ␤ 2 AR. The secondary decrease in the contraction rate was mediated by G i and could be blocked by pertussis toxin. Furthermore, a non-selective endocytosis inhibitor, concanavalin A, inhibited the internalization of wild type ␤ 2 AR and the mutated ␤ 1 AR-PDZ, and blocked the coupling of both receptors to G i . Finally, treating myocytes with a membrane-permeable peptide representing ␤ 1 AR PDZ motif caused the endogenous ␤ 1 AR to behave like ␤ 1 AR-PDZ. These studies suggest that association of the ␤ 1 AR with PSD-95 or a related protein dictates signaling specificity by retaining the receptor at the cell surface and preventing interaction with G i .␤ adrenergic receptors (␤ 1 AR, ␤ 2 AR, and ␤ 3 AR) 1 are heptahelical G protein-coupled receptors (GPCRs) that mediate physiological responses to the hormone epinephrine and the neurotransmitter norepinephrine. Both ␤ 1 and ␤ 2 ARs are expressed in the heart and play critical roles in regulating cardiac function in animals (1). In vitro studies show that ␤ 1 and ␤ 1 ARs have very similar signaling properties when expressed in undifferentiated cell lines (2). However, evidence also suggests that they have different signaling properties in differentiated cells in vivo (3, 4). Moreover, these receptors may differ in other functional parameters such as desensitization (5). ␤ 1 AR knockout mice lack the normal inotropic and chronotropic response to the adrenergic agonist, isoproterenol (6), whereas these responses are preserved in ␤ 2 AR knockout mice (7).We recently reported that ␤ 1 and ␤ 2 ARs regulate contraction rate in mouse neonatal myocytes through different signal transduction pathways (8). Activated ␤ 1 AR couples only to G s and leads to a PKA-dependent increase in contraction rate. Activated ␤ 2 AR undergoes sequential coupling to G s and G i and has a biphasic effect on contraction rate, with an initial PKAindependent increase followed by a sustained decrease that can be blocked by pertussis toxin (8). Recent biochemical evidence su...

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“…4). These results in mouse cardiac myocytes are unlike those reported in another study, which have implied that the WT ␤ 1 -AR did not internalize in mouse cardiac myocytes in response to isoproterenol (45). Our results clearly showed that the ␤ 1 -AR was efficiently trafficked in mouse cardiac myocytes.…”

Resistance of the Human β1-Adrenergic Receptor to Agonist-mediated Down-regulation

“…␤ 1 AR has no tyrosine residues in its C-tail but does terminate in a PDZ binding motif (ESKV) that is recognized by PSD-95 and MAGI-2, two postsynaptic scaffolding proteins that respectively inhibit and enhance receptor internalization (48,49). Mutation of the PDZ motif of mouse ␤ 1 AR enhances agonist-mediated endocytosis in mouse cardiac myocytes (50). When we used chimeric receptors in which the C-tails had been exchanged, we found that upon persistent agonist stimulation, ␤ 2 /␤ 1 ct-AR underwent up-regulation, and ␤ 1 /␤ 2 ct-AR underwent down-regulation, the order of down-regulation being ␤ 2 AR Ͼ ␤ 1 /␤ 2 ct-AR Ͼ Ͼ ␤ 2 /␤ 1 ct-AR Ͼ ␤ 1 AR.…”

Neurohumoral activation in heart failure: the role of adrenergic receptors

“…In neonatal cardiac myocytes from β 1 and β 2 receptor double knockout mice, dual coupling of the β 3 subtype to both G s and G i , with the G i component dominating, has been described (Devic et al 2001). These specific signaling properties of β receptor subtypes have been linked to subtype-selective association with intracellular scaffolding and signaling proteins, and distinct subcellular localization (Steinberg 1999, Hall and Lefkowitz 2002, Xiang et al 2002, Xiang and Kobilka 2003a. Figure 3A is a schematic representation of β-adrenergic receptor signaling in the heart.…”