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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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Norepinephrine inhibition of [3H]rauwolscine binding. The data points are the means ± SEM of three independent inhibition experiments, each done in duplicate. The concentration of [3H]rauwolscine was 0.12 ± 0.01 pM. The IC50 values determined from these data are 380 nM, 170 nM and 160 nM for the wild-type (SSTS), SSVA and AAVA receptors, respectively. The 100% values were 1223, 755 and 285 cpm for the wild-type (SSTS), SSVA and AAVA mutants, respectively. The means of the Ki values determined in the three individual experiments are given in Table 2.
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Figure 3: Norepinephrine inhibition of [3H]rauwolscine binding. The data points are the means ± SEM of three independent inhibition experiments, each done in duplicate. The concentration of [3H]rauwolscine was 0.12 ± 0.01 pM. The IC50 values determined from these data are 380 nM, 170 nM and 160 nM for the wild-type (SSTS), SSVA and AAVA receptors, respectively. The 100% values were 1223, 755 and 285 cpm for the wild-type (SSTS), SSVA and AAVA mutants, respectively. The means of the Ki values determined in the three individual experiments are given in Table 2.

Mentions: The results of [3H]rauwolscine saturation experiments using the wild-type and mutated receptors from two independent transfections of both mutations are given in Table 1. The Kd values for the two mutants (0.071–0.118 nM) were similar to those of the wild-type (0.113 nM) and similar to the previously reported Kd of 0.056 nM for the wild-type alpha-2 receptor for [3H]rauwolscine in NaPO4 buffer [23]. Inhibition experiments with [3H]rauwolscine indicated that the affinity of norepinephrine for the mutated receptors was approximately two-fold higher (lower Kd value) than for wild-type receptor (Figure 3).


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)

Norepinephrine inhibition of [3H]rauwolscine binding. The data points are the means ± SEM of three independent inhibition experiments, each done in duplicate. The concentration of [3H]rauwolscine was 0.12 ± 0.01 pM. The IC50 values determined from these data are 380 nM, 170 nM and 160 nM for the wild-type (SSTS), SSVA and AAVA receptors, respectively. The 100% values were 1223, 755 and 285 cpm for the wild-type (SSTS), SSVA and AAVA mutants, respectively. The means of the Ki values determined in the three individual experiments are given in Table 2.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Norepinephrine inhibition of [3H]rauwolscine binding. The data points are the means ± SEM of three independent inhibition experiments, each done in duplicate. The concentration of [3H]rauwolscine was 0.12 ± 0.01 pM. The IC50 values determined from these data are 380 nM, 170 nM and 160 nM for the wild-type (SSTS), SSVA and AAVA receptors, respectively. The 100% values were 1223, 755 and 285 cpm for the wild-type (SSTS), SSVA and AAVA mutants, respectively. The means of the Ki values determined in the three individual experiments are given in Table 2.
Mentions: The results of [3H]rauwolscine saturation experiments using the wild-type and mutated receptors from two independent transfections of both mutations are given in Table 1. The Kd values for the two mutants (0.071–0.118 nM) were similar to those of the wild-type (0.113 nM) and similar to the previously reported Kd of 0.056 nM for the wild-type alpha-2 receptor for [3H]rauwolscine in NaPO4 buffer [23]. Inhibition experiments with [3H]rauwolscine indicated that the affinity of norepinephrine for the mutated receptors was approximately two-fold higher (lower Kd value) than for wild-type receptor (Figure 3).

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