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A noninternalized nondesensitized truncated AT1A receptor transduces an amplified ANG II signal
S Conchon1, N Peltier, P Corvol
1Institut National de la Santé et de la Recherche Médicale Unité 36, Collège de France, Paris, France.
Abstract:
The structural determinants of the rat angiotensin (ANG) II AT1A receptor involved in receptor internalization, desensitization, and activation are investigated by producing six mutants that had progressively larger deletions of the cytoplasmic tail (-13, -19, -24, -31, -46, and -56 residues, respectively). After stable transfection of the cDNAs into Chinese hamster ovary cells, all mutants, except the most truncated, exhibit normal [Sar1]ANG II affinities [dissociation constant (Kd) = 0.19-0.70 nM] compared with the wild-type (WT) receptor (Kd = 0.62 nM) and are able to activate a Gq/11 protein and a phospholipase C as measured by the ANG II-induced inositol phosphate (IP) turnover in the different clones. However, one of these mutants, delta 329 (deletion of 31 residues), exhibits a peculiar phenotype. This mutant shows a reduced ligand-induced internalization as measured by the acid-washing procedure (only 32% of receptors are internalized vs. 83% for WT). Moreover, the delta 329 mutant is less desensitized by a pretreatment with either ANG II (15% desensitization of ANG II-stimulated IP turnover vs. 60% for WT receptor) or the phorbol ester phorbol 12-myristate 13-acetate (no desensitization vs. 29% for WT receptor). These functional modifications of the delta 329 mutant are associated with the transduction of an amplified signal as demonstrated on both IP turnover and an integrated physiological effect of ANG II. Taken together, these data indicate that the sequence 329SLSTKMS335 of the rat AT1A receptor is involved in both receptor internalization and desensitization. This is the first demonstration that a desensitization- and internalization-defective AT1A receptor mutant is also hyperreactive and mediates augmented cellular responses.
Insights
Researchers studied rat angiotensin II AT1A receptor mutants to understand internalization and desensitization. A specific mutant (delta 329) showed impaired internalization and desensitization, leading to amplified cellular responses.
Area of Science:
- Molecular Pharmacology
- Cell Signaling
- Receptor Biology
Background:
- The angiotensin II AT1A receptor plays a crucial role in cardiovascular regulation.
- Receptor internalization and desensitization are key mechanisms modulating cellular responses to angiotensin II.
- Understanding the structural basis of these processes is vital for developing targeted therapies.
Purpose of the Study:
- To identify structural determinants within the cytoplasmic tail of the rat AT1A receptor responsible for internalization and desensitization.
- To investigate the functional consequences of impaired internalization and desensitization on receptor signaling.
- To characterize a novel AT1A receptor mutant with altered desensitization and internalization properties.
Main Methods:
- Generation of six rat AT1A receptor mutants with progressive deletions in the cytoplasmic tail.
- Stable transfection into Chinese hamster ovary cells and assessment of [Sar1]ANG II binding affinities (Kd).
- Measurement of ANG II-induced inositol phosphate (IP) turnover, receptor internalization (acid-washing), and desensitization (ANG II or phorbol ester pretreatment).
Main Results:
- Most mutants retained normal ligand binding affinities and Gq/11 protein/phospholipase C activation.
- The delta 329 mutant (31 residue deletion) exhibited significantly reduced ligand-induced internalization (32% vs. 83% WT).
- Delta 329 displayed diminished desensitization to ANG II (15% vs. 60% WT) and phorbol ester (0% vs. 29% WT), coupled with amplified signaling.
Conclusions:
- The sequence 329SLSTKMS335 in the rat AT1A receptor cytoplasmic tail is critical for receptor internalization and desensitization.
- A mutant defective in desensitization and internalization (delta 329) demonstrates hyperreactivity and augmented cellular responses.
- This study provides the first evidence linking impaired AT1A receptor desensitization/internalization to enhanced signaling and cellular effects.