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?1B-adrenergic receptors differentially associate with Rab proteins during homologous and heterologous desensitization.

ABSTRACT: Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that ?1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing ?1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when ?1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient ?1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that ?1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs. heterologous).

SUBMITTER: Castillo-Badillo JA

PROVIDER: S-EPMC4370394 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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α1B-adrenergic receptors differentially associate with Rab proteins during homologous and heterologous desensitization.

Castillo-Badillo Jean A JA Sánchez-Reyes Omar B OB Alfonzo-Méndez Marco A MA Romero-Ávila M Teresa MT Reyes-Cruz Guadalupe G García-Sáinz J Adolfo JA

PloS one 20150323 3

Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, wh ...[more]

PMID: 25799564

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Project description:AimsCardiac ?-adrenergic receptor (?AR) signalling is susceptible to heterologous desensitization by different neurohormonal stimuli in clinical conditions associated with heart failure. We aim to examine the underlying mechanism of cross talk between ?ARs and a set of G-protein coupled receptors (GPCRs) activated by hormones/agonists.Methods and resultsRat ventricular cardiomyocytes were used to determine heterologous phosphorylation of ?ARs under a series of GPCR agonists. Activation of Gs-coupled dopamine receptor, adenosine receptor, relaxin receptor and prostaglandin E2 receptor, and Gq-coupled ?1 adrenergic receptor and angiotensin II type 1 receptor promotes phosphorylation of ?1AR and ?2AR at putative protein kinase A (PKA) phosphorylation sites; but activation of Gi-coupled ?2 adrenergic receptor and activation of protease-activated receptor does not. The GPCR agonists that promote ?2AR phosphorylation effectively inhibit ?AR agonist isoproterenol-induced PKA phosphorylation of phospholamban and contractile function in ventricular cardiomyocytes. Heterologous GPCR stimuli have minimal to small effect on isoproterenol-induced ?2AR activation and G-protein coupling for cyclic adenosine monophosphate (cAMP) production. However, these GPCR stimuli significantly promote phosphorylation of phosphodiesterase 4D (PDE4D), and recruit PDE4D to the phosphorylated ?2AR in a ?-arrestin 2 dependent manner without promoting ?2AR endocytosis. The increased binding between ?2AR and PDE4D effectively hydrolyzes cAMP signal generated by subsequent stimulation with isoproterenol. Mutation of PKA phosphorylation sites in ?2AR, inhibition of PDE4, or genetic ablation of PDE4D or ?-arrestin 2 abolishes this heterologous inhibitory effect. Ablation of ?-arrestin 2 or PDE4D gene also rescues ?-adrenergic stimuli-induced myocyte contractile function.ConclusionsThese data reveal essential roles of ?-arrestin 2 and PDE4D in a common mechanism for heterologous desensitization of cardiac ?ARs under hormonal stimulation, which is associated with impaired cardiac function during the development of pathophysiological conditions.

Project description:The number and activity of Cav1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca2+-induced Ca2+ release and myocardial contraction. β-Adrenergic receptor (βAR) activation stimulates sarcolemmal insertion of CaV1.2. This supplements the preexisting sarcolemmal CaV1.2 population, forming large "superclusters" wherein neighboring channels undergo enhanced cooperative-gating behavior, amplifying Ca2+ influx and myocardial contractility. Here, we determine this stimulated insertion is fueled by an internal reserve of early and recycling endosome-localized, presynthesized CaV1.2 channels. βAR-activation decreased CaV1.2/endosome colocalization in ventricular myocytes, as it triggered "emptying" of endosomal CaV1.2 cargo into the t-tubule sarcolemma. We examined the rapid dynamics of this stimulated insertion process with live-myocyte imaging of channel trafficking, and discovered that CaV1.2 are often inserted into the sarcolemma as preformed, multichannel clusters. Similarly, entire clusters were removed from the sarcolemma during endocytosis, while in other cases, a more incremental process suggested removal of individual channels. The amplitude of the stimulated insertion response was doubled by coexpression of constitutively active Rab4a, halved by coexpression of dominant-negative Rab11a, and abolished by coexpression of dominant-negative mutant Rab4a. In ventricular myocytes, βAR-stimulated recycling of CaV1.2 was diminished by both nocodazole and latrunculin-A, suggesting an essential role of the cytoskeleton in this process. Functionally, cytoskeletal disruptors prevented βAR-activated Ca2+ current augmentation. Moreover, βAR-regulation of CaV1.2 was abolished when recycling was halted by coapplication of nocodazole and latrunculin-A. These findings reveal that βAR-stimulation triggers an on-demand boost in sarcolemmal CaV1.2 abundance via targeted Rab4a- and Rab11a-dependent insertion of channels that is essential for βAR-regulation of cardiac CaV1.2.

Project description:1. Peptidergic neurones accumulate amines via an unusual uptake process, designated Transport-P. [(3)H]-prazosin binds to alpha(1) adrenoceptors on these cells and is displaceable by unlabelled prazosin in concentrations up to 10(-7) M. However, at greater concentrations of prazosin, there is a paradoxical accumulation of [(3)H]-prazosin which we have attributed to Transport-P. Uptake of prazosin via Transport-P is detectable at 10(-10) M prazosin concentration, is linear up to 10(-7) M and at greater concentrations becomes non-linear. In contrast, in noradrenergic neurones, noradrenaline uptake is linear and saturates above 10(-7) M. In noradrenergic neurones and in non-neuronal cells, there is no uptake of prazosin in concentrations up to 10(-6) M, suggesting that Transport-P is a specialised function of peptidergic neurones. 2. Using a mouse peptidergic (gonadotrophin-releasing hormone, GnRH) neuronal cell line which possesses Transport-P, we have studied the interaction of alpha(1) adrenoceptors with Transport-P. Polymerase chain reactions and DNA sequencing of the products demonstrated that only the alpha(1B) sub-type of adrenoceptors is present in GnRH cells. 3. In COS cells transfected with alpha(1b) adrenoceptor cDNA and in DDT(1) MF-2 cells which express native alpha(1B) adrenoceptors, [(3)H]-prazosin was displaced by unlabelled prazosin in a normal equilibrium process, with no prazosin paradox in concentrations up to 10(-6) M. In DDT(1) MF-2 cells, [(3)H]-prazosin was displaced likewise by a series of alpha(1) adrenergic agonists, none of which increased the binding of [(3)H]-prazosin. Hence, the prazosin paradox is not due to some function of alpha(1) adrenoceptors, such as internalization of ligand-receptor complexes. 4. In neurones which possess Transport-P, transfection with alpha(1b) adrenoceptor cDNA resulted in over-expression of alpha(1B) adrenoceptors, but the prazosin paradox was unaltered. Thus, alpha(1) adrenoceptors and Transport-P mediate distinct functions in peptidergic neurones.

Project description:Salmeterol is a long-acting ?2-adrenergic receptor (?2AR) agonist that is widely used as a bronchodilator for the treatment of persistent asthma and chronic obstructive pulmonary disease in conjunction with steroids. Previous studies demonstrated that salmeterol showed weak efficacy for activation of adenylyl cyclase; however, its efficacy in the complex desensitization of the ?2AR remains poorly understood. In this work, we provide insights into the roles played by the G protein-coupled receptor kinase/arrestin and protein kinase A in salmeterol-mediated desensitization through bioluminescence resonance energy transfer (BRET) studies of liganded-?2AR binding to arrestin and through kinetic studies of cAMP turnover. First, BRET demonstrated a much reduced efficacy for salmeterol recruitment of arrestin to ?2AR relative to isoproterenol. The ratio of BRETISO/BRETSALM after 5-minute stimulation was 20 and decreased to 5 after 35 minutes, reflecting a progressive decline in BRETISO and a stable BRETSALM. Second, to assess salmeterol efficacy for functional desensitization, we examined the kinetics of salmeterol-induced cAMP accumulation (0-30 minutes) in human airway smooth muscle cells in the presence and absence of phosphodiesterase inhibition. Analysis of shaping of cAMP turnover for both agonists demonstrated significant salmeterol desensitization, although it was reduced relative to isoproterenol. Using an isoproterenol rescue protocol after either short-term (10 minutes) or long-term (2 and 14 hours) salmeterol pretreatments, we found that salmeterol progressively depressed isoproterenol stimulation but did not prevent subsequent rescue by isoproterenol and additional isoproterenol-mediated desensitization. Our findings reveal a complex efficacy for functional desensitization, demonstrating that although salmeterol shows weak efficacy for adenylyl cyclase activation and G protein-coupled receptor kinase/arrestin-mediated desensitization, it acts as a strong agonist in highly amplified protein kinase A-mediated events.

Project description:The homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase induced by lutropin (LH) was characterized with the aid of forskolin and cholera toxin. Forskolin stimulated cyclic AMP production in a dose-dependent manner, with linear kinetics up to 2h. Forskolin also potentiated the action of LH on cyclic AMP production, but was only additive with cholera toxin. Preincubation of rat Leydig tumour cells with LH (1.0 micrograms/ml) for 1 h produced a desensitization of the subsequent LH (1.0 micrograms/ml)-stimulated cyclic AMP production, whereas the responses to cholera toxin (5.0 micrograms/ml), forskolin (100 microM), LH plus forskolin or cholera toxin plus forskolin were unaltered. In contrast, preincubation with LH for 20h produced a desensitization to all the stimuli tested. When rat Leydig tumour cells were preincubated for 1h with forskolin or dibutyryl cyclic AMP, the only subsequent response that was significantly altered was that to LH plus forskolin after preincubation with forskolin. However, preincubation for 20h with forskolin or dibutyryl cyclic AMP induced a desensitization to all stimuli subsequently tested. LH produced a rapid (0-1h) homologous desensitization, which was followed by a slower (2-8h)-onset heterologous desensitization. Forskolin and dibutyryl cyclic AMP were only able to induce heterologous desensitization. The rate of desensitization induced by either forskolin or dibutyryl cyclic AMP was similar to the rate of heterologous desensitization induced by LH. These results demonstrate that in purified rat Leydig tumour cells LH produces an initial homologous desensitization of adenylate cyclase that involves a cyclic AMP-independent lesion at or proximal to the guanine nucleotide regulatory protein (G-protein). This is followed by heterologous desensitization, which can also be induced by forskolin or dibutyryl cyclic AMP, thus indicating that LH-induced heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase involves a cyclic AMP-dependent lesion that is after the G-protein.

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?1B-adrenergic receptors differentially associate with Rab proteins during homologous and heterologous desensitization.

Publications

α1B-adrenergic receptors differentially associate with Rab proteins during homologous and heterologous desensitization.

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