Apparent Loss-of-Function Mutant GPCRs Revealed as Constitutively Desensitized Receptors

Abstract

The DRY motif is a triplet amino acid sequence (aspartic acid, arginine, and tyrosine) that is highly conserved in G protein-coupled receptors (GPCRs). Recently, we have shown that a molecular determinant for nephrogenic diabetes insipidus, the vasopressin receptor with a substitution at the DRY motif arginine (V2R R137H), is a constitutively desensitized receptor that is unable to couple to G proteins due to its constitutive association with beta-arrestin [Barak, L. S. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 93-98]. Additionally, the mutant receptors are localized in endocytic vesicles, identical to wild-type receptors stimulated with agonist. In this study, we asked whether the constitutively desensitized phenotype observed in the V2R R137H represents a general paradigm that may be extended to other GPCRs. We show that arginine substitutions in the DRY motifs of the alpha(1B) adrenergic receptor (alpha(1B)-AR) and angiotensin II type 1A receptor (AT(1A)R) result in receptors that are uncoupled from G proteins, associated with beta-arrestins, and found localized in endocytic vesicles rather than at the plasma membrane in the absence of agonists. The localization of the alpha(1B)-ARs and AT(1A)Rs with arginine substitutions can be restored to the plasma membrane by either using selective antagonists or preventing the endocytosis of the beta-arrestin-receptor complexes. These results indicate that the arginine residue of the DRY motif is essential for preserving the localization of the inactive receptor complex. Furthermore, constitutive desensitization may underlie some loss-of-function receptor phenotypes and represent an unappreciated mechanism of hormonal resistance.

Identification of Drosophila Neuropeptide Receptors by G Protein-coupled Receptors-β-Arrestin2 Interactions

“…We reasoned that this lack of a detectable response to Ast-C might be due to intrinsic phosphorylation by the endogenous complement of GRKs. In mammalian GPCRs, certain receptors are constitutively phosphorylated and associated with ␤-arrestin which, at steady state, results in their trafficking to endocytic vesicles and a loss of signaling function (16,23). With these receptors, inhibiting clathrin-mediated endocytosis reverses their intracellular localization phenotype.…”

“…However, certain GPCRs, such as constitutively active and "loss of function" mutant receptors, are efficient GRK substrates even in the absence of agonist activation (16,23). This property leads to the constitutive trafficking of these receptors to endocytic vesicles and thus effectively removes them from the plasma membrane and inhibits their signaling function.…”

“…This property leads to the constitutive trafficking of these receptors to endocytic vesicles and thus effectively removes them from the plasma membrane and inhibits their signaling function. Trapping them at the plasma membrane using internalization inhibitors such as dynamin K44A permits the visualization of these arrestin-receptor complexes (16,23). We found that overexpression of dynamin K44A did not alter the normal ␤arr-2 translocation response profile of the proctolin receptor CG6986.…”

“…Membranes were resuspended in assay buffer (50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 5 mM MgCl 2 , 1 mM EDTA, 1 mM dithiothreitol, 10 M GDP). Membranes (20 g of protein per assay tube) were incubated in the presence or absence of DMS and 100 pM [ 35 S]GTP␥S (1250 Ci/mmol, PerkinElmer Life Sciences) for 1 h at 30°C as described previously (16). Binding was terminated by rapid filtration over GF/B filters using a Brandel cell harvester (Brandel, Gaithersburg, MD).…”

Effect of torsinA on membrane proteins reveals a loss of function and a dominant-negative phenotype of the dystonia-associated ΔE-torsinA mutant

“…The amino-terminal GFP-tagged DAT (GFP-DAT) was generously provided by S. G. Amara (University of Pittsburgh). The GFPtagged ␣1B (␣1BAR-GFP) and ␤2 adrenergic receptors (␤2AR-GFP) and D2DAR-GFP have been described (20,21). The human norepinephrine transporter was subcloned into the mammalian expression vector pcDNA3.1.…”