Fuguo Zhou

Anterograde Trafficking of G Protein-Coupled Receptors: Function of the C-Terminal F(X)6LL Motif in Export from the Endoplasmic Reticulum

We have reported previously that the F(X)6LL motif in the C termini is essential for export of α2B-adrenergic (α2B-AR) and angiotensin II type 1 receptors (AT1Rs) from the endoplasmic reticulum (ER). Here, we further demonstrate that mutation of the F(X)6LL motif similarly abolished the cell-surface expression of α2B-AR, AT1R, α1B-AR, and β2-AR, suggesting that the F(X)6LL motif plays a general role in ER export of G protein-coupled receptors (GPCRs). Mutation of Phe to Val, Leu, Trp, and Tyr, and mutation of LL to FF and VV, markedly inhibited α2B-AR transport, indicating that the F(X)6LL function cannot be fully substituted by other hydrophobic residues. The structural analysis revealed that the Phe residue in the F(X)6LL motif is buried in the transmembrane domains and possibly interacts with Ile58 in β2-AR and Val42 in α2B-AR, whereas the LL motif is exposed to the cytosolic space. Indeed, mutation of Ile58 in β2-AR and Val42 in α2B-AR markedly disrupted cell surface transport of the receptors. It is noteworthy that the Val and Ile residues are highly conserved among the GPCRs carrying the F(X)6LL motif. Furthermore, the Phe mutant exhibited a stronger interaction with ER chaperones and was more potently rescued by physical and chemical treatments than the LL mutant. These data suggest that the Phe residue is probably involved in folding of α2B-AR and β2-AR, possibly through interaction with other hydrophobic residues in neighboring domains. These data also provide the first evidence implying crucial roles of the C termini possibly through modulating multiple events in anterograde trafficking of GPCRs.

Enhancement of the Recycling and Activation of β-Adrenergic Receptor by Rab4 GTPase in Cardiac Myocytes

We investigate the role of Rab4, a Ras-like small GTPase coordinating protein transport from the endosome to the plasma membrane, on the recycling and activation of endogenous β-adrenergic receptor (β-AR) in HL-1 cardiac myocytes in vitro and transgenic mouse hearts in vivo. β1-AR, the predominant subtype of β-AR in HL-1 cardiac myocytes, was internalized after stimulation with isoproterenol (ISO) and fully recycled at 4 h upon ISO removal. Transient expression of Rab4 markedly facilitated recycling of internalized β-AR to the cell surface and enhanced β-AR signaling as measured by ISO-stimulated cAMP production. Transgenic overexpression of Rab4 in the mouse myocardium significantly increased the number of β-AR in the plasma membrane and augmented cAMP production at the basal level and in response to ISO stimulation. Rab4 overexpression induced concentric cardiac hypertrophy with a moderate increase in ventricle/body weight ratio and posterior wall thickness and a selective up-regulation of the β-myosin heavy chain gene. These data provide the first evidence indicating that Rab4 is a rate-limiting factor for the recycling of endogenous β-AR and augmentation of Rab4-mediated traffic enhances β-AR function in cardiac myocytes.

Differential regulation of the cell-surface targeting and function of β- and α1-adrenergic receptors by Rab1 GTPase in cardiac myocytes

The molecular mechanism underlying the export from the endoplasmic reticulum (ER) to the cell surface and its role in the regulation of signaling of adrenergic receptors (ARs) remain largely unknown. In this report, we determined the role of Rab1, a Ras-like GTPase that coordinates protein transport specifically from the ER to the Golgi, in the cell surface targeting and function of endogenous β- and α1-ARs in neonatal rat ventricular myocytes. Adenovirus-driven expression of Rab1 into myocytes selectively increased the cell-surface number of α1-AR, but not β-AR, whereas the dominant-negative mutant Rab1N124I significantly reduced the cell-surface expression of β-AR and α1-AR. Brefeldin A inhibited β-AR and α1-AR export and antagonized the Rab1 effect on α1-AR expression. Manipulation of Rab1 function similarly influenced the transport of α1A- and α1B-ARs as well as β1- and β2-ARs. Fluorescent microscopy analysis demonstrated that expression of Rab1N124I and Rab1 small interfering RNA induced a marked accumulation of GFP-tagged β2-AR and α1B-AR in the ER. Consistent with the effects on receptor cell-surface targeting, Rab1 selectively enhanced ERK1/2 activation and hypertrophic growth in response to the α1-AR agonist phenylephrine but not to the β-AR agonist isoproterenol. Rab1N124I inhibited both agonist-mediated ERK1/2 activation and hypertrophic growth in neonatal myocytes. These results demonstrate that the cell-surface targeting and signaling of β- and α1-ARs require Rab1 and are differentially modulated by augmentation of Rab1 function. Our data provide strong evidence implicating the ER-to-Golgi traffic as a site for selective manipulation of distinct AR function in cardiac myocytes.

Endoplasmic reticulum export of adrenergic and angiotensin II receptors is differentially regulated by Sar1 GTPase

The molecular mechanism underlying the export of G protein-coupled receptors (GPCRs) from the endoplasmic reticulum (ER) remains largely unknown. In this manuscript, we investigated the role of Sar1 GTPase, which coordinates the assembly and budding of COPII-coated vesicles, in the cell-surface targeting, signaling and ER export of alpha(2B)-adrenergic (alpha(2B)-AR), beta(2)-AR and angiotensin II type 1 receptors (AT1R). The cell-surface expression of alpha(2B)-AR, beta(2)-AR and AT1R, and receptor-mediated ERK1/2 activation were significantly attenuated by the GTP-bound mutant Sar1H79G, suggesting that export from the ER of these receptors is mediated through the Sar1-dependent COPII-coated vesicles. Interestingly, subcellular distribution analyses showed that alpha(2B)-AR and AT1R were highly concentrated at discrete locations near the nucleus in cells expressing Sar1H79G, whereas beta(2)-AR exhibited an ER distribution. These data indicate that Sar1-catalyzed efficient GTP hydrolysis differentially regulates ER export of adrenergic and angiotensin II receptors. These data provide the first evidence indicating distinct mechanisms for the recruitment of different GPCRs into the COPII vesicles on the ER membrane.

ADP-ribosylation factors modulate the cell surface transport of G protein-coupled receptors

ADP-ribosylation factors (ARFs) regulate vesicular traffic through recruiting coat proteins. However, their functions in the anterograde transport of nascent G protein-coupled receptors (GPCRs) from the endoplasmic reticulum to the plasma membrane remain poorly explored. Here we show that treatment with brefeldin A, an inhibitor of guanine nucleotide exchange on ARFs, markedly attenuated the cell surface numbers of α2B-adrenergic receptor (AR), β2-AR, angiotensin II type 1 receptor, and chemokine (CXC motif) receptor 4. Functional inhibition of individual ARF GTPases by transient expression of the GDP-bound, GTP-bound, and guanine nucleotide-deficient mutants showed that the five human ARFs differentially modulated receptor cell surface expression and that the ARF1 mutants produced the most profound inhibitory effect. Furthermore, expression of the ARF1 GTPase-activating protein (GAP) ARFGAP1 significantly blocked receptor transport. Interestingly, the GDP- and GTP-bound ARF1 mutants arrested the receptors in distinct intracellular compartments. Consistent with the reduced receptor cell surface expression, extracellular signal-regulated kinase 1 and 2 activation by receptor agonists was significantly attenuated by the GDP-bound mutant ARF1T31N. Moreover, coimmunoprecipitation showed that α2B-AR associated with ARF1 and glutathione transferase pull-down assay indicated that the α2B-AR C terminus directly interacted with ARF1. These data show that ARF1 GTPase is involved in the regulation of cell surface expression of GPCRs at multiple transport steps.

Analysis of Rab1 Function in Cardiomyocyte Growth

Protein transport between intracellular organelles is coordinated by Rab GTPases. As an initial approach to defining the function of Rab GTPases in cardiomyocytes, our laboratory focused on Rab1, which regulates protein transport specifically from the endoplasmic reticulum (ER) to the Golgi apparatus. Our studies have demonstrated that adenovirus-driven expression of Rab1 promotes cell growth of primary cultures of neonatal cardiomyocytes in vitro and that transgenic expression of Rab1 in the myocardium induces cardiac hypertrophy in mouse hearts in vivo. These data provide strong evidence implicating that ER-to-Golgi protein transport functions as a regulatory site for control of cardiomyocyte growth. Here we describe a sets of methods used in our laboratory to characterize the function of Rab1 GTPase in modulating cardiac myocyte growth.