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Down-regulation of the alpha-2C adrenergic receptor: involvement of a serine/threonine motif in the third cytoplasmic loop.

Deupree JD, Borgeson CD, Bylund DB - BMC Pharmacol. (2002)

Bottom Line: Interestingly, the human alpha-2C receptor does not down-regulate, whereas the opossum alpha-2C receptor does down-regulate.These results indicate that a motif with four hydroxyl amino acid residues in an acidic environment is important for down-regulation of the opossum alpha-2C adrenergic receptor.Because these are potential GRK phosphorylation sites, we suggest that GRK phosphorylation may be involved in alpha-2C adrenergic receptor down-regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Nebraska Medical Center, Omaha, NE 68198-6260, USA. jddeupre@unmc.edu

ABSTRACT

Background: The mechanisms by which alpha-2 adrenergic receptors are down-regulated following chronic exposure to agonist are not well understood. Interestingly, the human alpha-2C receptor does not down-regulate, whereas the opossum alpha-2C receptor does down-regulate. A comparison of the amino acid sequence of the third intracellular loop of these two receptors shows that the opossum alpha-2C receptor contains a potential G protein-coupled receptor kinase (GRK)phosphorylation motif (EESSTSE) with four hydroxyl residues, whereas the human alpha-2C receptor motif only contains two hydroxyl residues (DESSAAAAE). Because a similar acidic serine-rich motif (EESSSSD) in the human alpha-2 adrenergic receptor has been demonstrated to be phosphorylated by GRK and all four serines are required for desensitization of the receptor, we sought to determine whether the EESSTSE sequence was involved in the down-regulation of the alpha-2C adrenergic receptor.

Results: Site-directed mutagenesis was used to mutate the opossum alpha-2C receptor to SSVA and AAVA in place of the SSTS wild-type sequence. Down-regulation experiments on CHO cells transfected with the receptors demonstrated that neither of the mutated receptors down-regulated following 24 h exposure to norepinephrine, whereas the wild-type receptor down-regulated to 65 +/- 10% of the control.

Conclusions: These results indicate that a motif with four hydroxyl amino acid residues in an acidic environment is important for down-regulation of the opossum alpha-2C adrenergic receptor. Because these are potential GRK phosphorylation sites, we suggest that GRK phosphorylation may be involved in alpha-2C adrenergic receptor down-regulation.

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The sequence of the third intracellular loop of the opossum alpha-2C adrenergic receptor. The sequence of the two mutated receptors created by site-directed mutagenesis are indicated. Potential phosphorylation sites (serine and threonine residues) are shown in red.
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Figure 1: The sequence of the third intracellular loop of the opossum alpha-2C adrenergic receptor. The sequence of the two mutated receptors created by site-directed mutagenesis are indicated. Potential phosphorylation sites (serine and threonine residues) are shown in red.

Mentions: The human alpha-2A adrenergic receptor has an acidic serine-rich motif (EESSSSD) in the third intracellular loop that has been shown to be important in agonist-induced desensitization but not in agonist-induced sequestration/internalization. Site-directed mutagenesis studies indicate that all four of these serines are phosphorylated by GRK, and all four are needed for desensitization [12]. The influence of this motif on down-regulation was not investigated, however. The opossum alpha-2C adrenergic receptor has a very similar acidic serine motif (EESSTSE) in the third intracellular loop (Figure 1; [21]), whereas the human alpha-2C receptor has a DESSAAAE sequence in the same region (Figure 2; [22]). Thus, we postulated that the lack of down-regulation in the human alpha-2C was related to having only two rather than four serines in the motif, and furthermore that the ability of the opossum alpha-2C to undergo down-regulation was dependent on having all four serine/threonine residues in the EESSTSE motif. To test this hypothesis, we mutated the opossum alpha-2C serines and threonine residues to alanines and valine, respectively, and tested whether the expressed receptors would down-regulate. Down-regulation was not observed in the mutated receptors following treatment with norepinephrine for 24 h.


Down-regulation of the alpha-2C adrenergic receptor: involvement of a serine/threonine motif in the third cytoplasmic loop.

Deupree JD, Borgeson CD, Bylund DB - BMC Pharmacol. (2002)

The sequence of the third intracellular loop of the opossum alpha-2C adrenergic receptor. The sequence of the two mutated receptors created by site-directed mutagenesis are indicated. Potential phosphorylation sites (serine and threonine residues) are shown in red.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC103669&req=5

Figure 1: The sequence of the third intracellular loop of the opossum alpha-2C adrenergic receptor. The sequence of the two mutated receptors created by site-directed mutagenesis are indicated. Potential phosphorylation sites (serine and threonine residues) are shown in red.
Mentions: The human alpha-2A adrenergic receptor has an acidic serine-rich motif (EESSSSD) in the third intracellular loop that has been shown to be important in agonist-induced desensitization but not in agonist-induced sequestration/internalization. Site-directed mutagenesis studies indicate that all four of these serines are phosphorylated by GRK, and all four are needed for desensitization [12]. The influence of this motif on down-regulation was not investigated, however. The opossum alpha-2C adrenergic receptor has a very similar acidic serine motif (EESSTSE) in the third intracellular loop (Figure 1; [21]), whereas the human alpha-2C receptor has a DESSAAAE sequence in the same region (Figure 2; [22]). Thus, we postulated that the lack of down-regulation in the human alpha-2C was related to having only two rather than four serines in the motif, and furthermore that the ability of the opossum alpha-2C to undergo down-regulation was dependent on having all four serine/threonine residues in the EESSTSE motif. To test this hypothesis, we mutated the opossum alpha-2C serines and threonine residues to alanines and valine, respectively, and tested whether the expressed receptors would down-regulate. Down-regulation was not observed in the mutated receptors following treatment with norepinephrine for 24 h.

Bottom Line: Interestingly, the human alpha-2C receptor does not down-regulate, whereas the opossum alpha-2C receptor does down-regulate.These results indicate that a motif with four hydroxyl amino acid residues in an acidic environment is important for down-regulation of the opossum alpha-2C adrenergic receptor.Because these are potential GRK phosphorylation sites, we suggest that GRK phosphorylation may be involved in alpha-2C adrenergic receptor down-regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Nebraska Medical Center, Omaha, NE 68198-6260, USA. jddeupre@unmc.edu

ABSTRACT

Background: The mechanisms by which alpha-2 adrenergic receptors are down-regulated following chronic exposure to agonist are not well understood. Interestingly, the human alpha-2C receptor does not down-regulate, whereas the opossum alpha-2C receptor does down-regulate. A comparison of the amino acid sequence of the third intracellular loop of these two receptors shows that the opossum alpha-2C receptor contains a potential G protein-coupled receptor kinase (GRK)phosphorylation motif (EESSTSE) with four hydroxyl residues, whereas the human alpha-2C receptor motif only contains two hydroxyl residues (DESSAAAAE). Because a similar acidic serine-rich motif (EESSSSD) in the human alpha-2 adrenergic receptor has been demonstrated to be phosphorylated by GRK and all four serines are required for desensitization of the receptor, we sought to determine whether the EESSTSE sequence was involved in the down-regulation of the alpha-2C adrenergic receptor.

Results: Site-directed mutagenesis was used to mutate the opossum alpha-2C receptor to SSVA and AAVA in place of the SSTS wild-type sequence. Down-regulation experiments on CHO cells transfected with the receptors demonstrated that neither of the mutated receptors down-regulated following 24 h exposure to norepinephrine, whereas the wild-type receptor down-regulated to 65 +/- 10% of the control.

Conclusions: These results indicate that a motif with four hydroxyl amino acid residues in an acidic environment is important for down-regulation of the opossum alpha-2C adrenergic receptor. Because these are potential GRK phosphorylation sites, we suggest that GRK phosphorylation may be involved in alpha-2C adrenergic receptor down-regulation.

Show MeSH
Related in: MedlinePlus