Dependence of agonist activation on a conserved apolar residue in the third intracellular loop of the AT1 angiotensin receptor

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 93; no. 19; pp. 10040 - 10045
• Main Authors: Hunyady, L, Zhang, M, Jagadeesh, G, Bor, M, Balla, T, Catt, K J
• Format: Journal Article
• Language: English
• Published: United States National Acad Sciences 17.09.1996; National Academy of Sciences
• Subjects: Amino Acid Sequence; Amino acids; Angiotensin II - metabolism; Angiotensin II - pharmacology; Animals; Biochemistry; Cell Membrane - metabolism; Cercopithecus aethiops; Conserved Sequence; COS Cells; Cytoplasm - metabolism; Endocytosis; Inositol Phosphates - metabolism; Kinetics; Lysine; Molecular Sequence Data; Muscle, Smooth - metabolism; Mutagenesis, Site-Directed; Protein Structure, Secondary; Proteins; Rats; Receptor, Angiotensin, Type 1; Receptors, Angiotensin - biosynthesis; Receptors, Angiotensin - chemistry; Receptors, Angiotensin - physiology; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Rodents; Sequence Deletion; Sequence Homology, Amino Acid; Transfection
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.19.10040

The coupling of agonist-activated seven transmembrane domain receptors to G proteins is known to involve the amino-terminal region of their third cytoplasmic loop. Analysis of the amino acids in this region of the rat type in angiotensin (AT1a) receptor identified Leu-222 as an essential residue in receptor activation by the physiological agonist, angiotensin II (Ang II). Nonpolar replacements for Leu-222 yielded functionally intact AT1 receptors, while polar or charged residues caused progressive impairment of Ang II-induced inositol phosphate generation. The decrease in agonist-induced signal generation was associated with a parallel reduction of receptor internalization, and was most pronounced for the Lys-222 mutant receptor. Although this mutant showed normal binding of the peptide antagonist, [Sar1,Ile6]Ang II, its affinity for Ang II was markedly reduced, consistent with its inability to adopt the high-affinity conformation. A search revealed that many Gq-coupled receptors contain an apolar amino acid (frequently leucine) in the position corresponding to Leu-222 of the AT1 receptor. These findings suggest that such a conserved apolar residue in the third intracellular loop is a crucial element in the agonist-induced activation of the AT1 and possibly many other G protein-coupled receptors.