Abstract
The role of conserved amino acids in the molecular mechanism(s) of activation of G protein-coupled receptors (GPCRs) was analyzed in mutant and wild-type rat AT angiotensin receptors expressed in COS-7 cells. The highly conserved Asp-Arg-Tyr (DRY) sequence is located at the ami no-terminal end of the second intracellular loop in position 125-127 of the AT, receptor. Individual alanine replacements of these residues had little effect on receptor function, but double alanine replacement of the Asp125 and Arg126 residues abolished inositol phosphate signaling. The Tyr215 residue of the AT, receptor in helix V at the amino-terminal portion of the third cytoplasmic loop is also conserved in most GPCRs. Substitution of this residue with phenylalanine or valine resulted in an activation-deficient AT, receptor. However, polar substituents such as asparagine, serine, and histidine did not attenuate receptor activation, suggesting that Tyr215 participates in a polar interaction, such as hydrogen bonding, with another part of the receptor molecule during activation. Since these regions of the second and third intracellular loops might be in molecular proximity, we tested the hypothesis that Tyr215 interacts with the Asp-Arg-Tyr sequence. Our results demonstrated that substitution of Arg127 in the Asp-Arg-Tyr sequence with phenylalanine partially restored angiotensin-stimulated inositol phosphate signal generation and internal ization of the activation-deficient Y215F mutant receptor. The effects of this gain-of-function mutation suggest that an intramolecular interaction between these regions is required for agonist-induced activation of the AT, receptor.
| Original language | English |
|---|---|
| Journal | FASEB Journal |
| Volume | 12 |
| Issue number | 8 |
| Publication status | Published - 1998 |
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ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Cell Biology
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An intramolecular interaction between conserved amino acids is required for activation of the angiotensin at, receptor. / Hunyady, L.; Zhang, M.; Gáborik, Z.; Catt, K. J.
In: FASEB Journal, Vol. 12, No. 8, 1998.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - An intramolecular interaction between conserved amino acids is required for activation of the angiotensin at, receptor
AU - Hunyady, L.
AU - Zhang, M.
AU - Gáborik, Z.
AU - Catt, K. J.
PY - 1998
Y1 - 1998
N2 - The role of conserved amino acids in the molecular mechanism(s) of activation of G protein-coupled receptors (GPCRs) was analyzed in mutant and wild-type rat AT angiotensin receptors expressed in COS-7 cells. The highly conserved Asp-Arg-Tyr (DRY) sequence is located at the ami no-terminal end of the second intracellular loop in position 125-127 of the AT, receptor. Individual alanine replacements of these residues had little effect on receptor function, but double alanine replacement of the Asp125 and Arg126 residues abolished inositol phosphate signaling. The Tyr215 residue of the AT, receptor in helix V at the amino-terminal portion of the third cytoplasmic loop is also conserved in most GPCRs. Substitution of this residue with phenylalanine or valine resulted in an activation-deficient AT, receptor. However, polar substituents such as asparagine, serine, and histidine did not attenuate receptor activation, suggesting that Tyr215 participates in a polar interaction, such as hydrogen bonding, with another part of the receptor molecule during activation. Since these regions of the second and third intracellular loops might be in molecular proximity, we tested the hypothesis that Tyr215 interacts with the Asp-Arg-Tyr sequence. Our results demonstrated that substitution of Arg127 in the Asp-Arg-Tyr sequence with phenylalanine partially restored angiotensin-stimulated inositol phosphate signal generation and internal ization of the activation-deficient Y215F mutant receptor. The effects of this gain-of-function mutation suggest that an intramolecular interaction between these regions is required for agonist-induced activation of the AT, receptor.
AB - The role of conserved amino acids in the molecular mechanism(s) of activation of G protein-coupled receptors (GPCRs) was analyzed in mutant and wild-type rat AT angiotensin receptors expressed in COS-7 cells. The highly conserved Asp-Arg-Tyr (DRY) sequence is located at the ami no-terminal end of the second intracellular loop in position 125-127 of the AT, receptor. Individual alanine replacements of these residues had little effect on receptor function, but double alanine replacement of the Asp125 and Arg126 residues abolished inositol phosphate signaling. The Tyr215 residue of the AT, receptor in helix V at the amino-terminal portion of the third cytoplasmic loop is also conserved in most GPCRs. Substitution of this residue with phenylalanine or valine resulted in an activation-deficient AT, receptor. However, polar substituents such as asparagine, serine, and histidine did not attenuate receptor activation, suggesting that Tyr215 participates in a polar interaction, such as hydrogen bonding, with another part of the receptor molecule during activation. Since these regions of the second and third intracellular loops might be in molecular proximity, we tested the hypothesis that Tyr215 interacts with the Asp-Arg-Tyr sequence. Our results demonstrated that substitution of Arg127 in the Asp-Arg-Tyr sequence with phenylalanine partially restored angiotensin-stimulated inositol phosphate signal generation and internal ization of the activation-deficient Y215F mutant receptor. The effects of this gain-of-function mutation suggest that an intramolecular interaction between these regions is required for agonist-induced activation of the AT, receptor.
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M3 - Article
AN - SCOPUS:33749095732
VL - 12
JO - FASEB Journal
JF - FASEB Journal
SN - 0892-6638
IS - 8
ER -