Dependence of agonist activation on a conserved apolar residue in the third intracellular loop of the AT1 angiotensin receptor.
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.
Hunyady
L
L
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Zhang
M
M
Jagadeesh
G
G
Bor
M
M
Balla
T
T
Catt
K J
KJ
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D003593:Cytoplasm / Q000378:metabolism; D004705:Endocytosis; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D051381:Rats; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D007295:Inositol Phosphates; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D011994:Recombinant Proteins; D000804:Angiotensin II; D008239:Lysine