Role of the Amino Terminus of the Third Intracellular Loop in Agonist-Promoted Downregulation of the α_2A-Adrenergic Receptor

Abstract

A prominent feature of long-term regulation of the alpha2A-adrenergic receptor (alpha2AAR) is a loss of cellular receptors over time (downregulation). The molecular determinants of downregulation were sought by targeting regions of the receptor involved in G protein coupling and phosphorylation. Mutated receptors, consisting of chimeric substitutions of analogous beta2-adrenergic receptor (beta2AR) and serotonin 5-hydroxytryptamine1A (5-HT1A) receptor sequence into the second intracellular loop (ICL2) (residues 113-149), the amino terminus (residues 218-235) and carboxy terminus (residues 355-371) of ICL3, and a deletion of the beta-adrenergic receptor kinase (betaARK) phosphorylation sites in the third intracellular loop (ICL3) (residues 293-304), were expressed in Chinese hamster ovary (CHO) cells. Wild-type alpha2AAR underwent 31% +/- 3% downregulation after 24 h of exposure to 100 microM epinephrine. Loss of downregulation was observed with some mutants, but this was not related to functional coupling to inhibitory or stimulatory guanine nucleotide regulatory binding proteins (Gi or GS) or to phosphorylation. Rather, any mutant with a substitution of the amino terminus of ICL3 (regardless of whether the substitution was with beta2AR or 5-HT1A sequence) resulted in upregulation. Studies with an inhibitor of protein synthesis indicated that the primary mechanism of downregulation of the alpha2AAR is agonist-promoted degradation of receptor protein which requires a destabilization sequence in the amino terminus of ICL3. Thus, in contrast to other G protein-coupled receptors, in which G protein coupling or phosphorylation are critical for long-term agonist regulation, the alpha2AAR has a specific structural domain distinct from these other functional regions that serves to direct agonist-promoted downregulation.

Up-Regulation of α1B-Adrenergic Receptors with Defects in G Protein Coupling: Ligand-Induced Protection from Receptor Instability

“…down-regulation (Jewell-Motz et al, 1997), similar to our discovery of the up-regulation of the ⌬12 and ⌬5 ␣ 1B -ARs during studies of down-regulation (Wang et al, 2002). Most of the other reports of up-regulation and instability originated from studies of constitutively active mutated receptors, including thyrotropin-releasing hormone receptors (Heinflink et al, 1995), ␤ 2 -ARs (Gether et al, 1997;Samama et al, 1997;Rasmussen et al, 1999), ␣ 2 -ARs (Betuing et al, 1997), ␣ 1B -ARs (Lee et al, 1997), and H 2 histamine receptors (Smit et al, 1996;Alewijnse et al, 1998).…”

“…However, it should be noted that protease inhibitors were routinely included in all incubations in those studies, and thus any protease-mediated instability mechanisms would have been missed. In contrast, receptor up-regulation in intact cells did seem to be accompanied by a corresponding increase in receptor protein assessed by Western blotting in one study with a mutated ␣ 2A -AR (Jewell-Motz et al, 1997) and assessed by fluorescence in one study with wild-type and mutated green fluorescent protein-tagged ␣ 1B -ARs (Stevens et al, 2000). The nature of the specific protease(s) involved in the instability of the ⌬12 ␣ 1B -AR, and whether the receptor itself or another associated protein is the relevant substrate, both remain to be determined.…”

“…This idea arises from a study of ␣ 2A -AR up-and down-regulation, in which a series of constructs with alterations in the N-terminal portion of the third intracellular loop were all found to exhibit up-regulation rather than down-regulation, in spite of the fact that some of these receptors were defective in coupling to G i or G s , whereas others exhibited normal coupling (Jewell-Motz et al, 1997). The failure of our ⌬[246 -261] ␣ 1B -AR mutation near the center of the third intracellular loop to confer up-regulation in spite of its uncoupling from G protein activation provides evidence for this model.…”

Involvement of G Protein-Coupled Receptor Kinase (GRK) 3 and GRK2 in Down-Regulation of the α2B-Adrenoceptor

“…The human ␣ 2B -AR polymorph (Del 301-303) lacking three glutamate residues in the third intracellular loop does not desensitize, and its long-term regulation was not tested (Small et al, 2001). The human ␣ 2A -AR mutant (Del 293-304) that lacks the four serine residues in the third intracellular loop thought to be phosphorylated by GRKs does not acutely desensitize but does undergo down-regulation (Jewell-Motz et al, 1997). In contrast to these observations, recent studies in BE(2)-C and BN17 cells and in GRK3-and GRK2-overexpressing NG108 cells suggest that the ␣ 2A -and ␣ 2B -AR are rendered more sensitive to agonist-induced down-regulation because of a 2-to 3-fold up-regulation of GRK3 (Bawa et al, 2003;Desai et al, 2004Desai et al, , 2005.…”

“…Mutation of potential GRK phosphorylation sites in ␣ 2C -AR blocks the agonistinduced down-regulation of these receptors expressed in opossum kidney cells (Deupree et al, 2002). When heterologously expressed in Chinese hamster ovary (CHO) cells, the ␣ 2B -AR (Jewell-Motz and Liggett, 1995) and other ␣ 2 -AR subtypes down-regulate in response to agonist exposure by mechanisms not dependent on GRK-mediated receptor phosphorylation (Jewell-Motz et al, 1997), but rather dependent on structural features such as palmitoylation (Eason et al, 1994). Ambiguity about the role of GRKs in receptor downregulation is not restricted to the ␣ 2 -AR but is observed for other GPCRs as well.…”