Structure/Function Relationships of a G-Protein Coupling Pocket Formed by the Third Intracellular Loop of the m5 Muscarinic Receptor†

Biochemistry ◽  
1998 ◽  
Vol 37 (12) ◽  
pp. 4052-4058 ◽  
Author(s):  
Ethan S. Burstein ◽  
Tracy A. Spalding ◽  
Mark R. Brann
Keyword(s):  
Structure Function ◽  
G Protein ◽  
Muscarinic Receptor ◽  
Intracellular Loop ◽  
G Protein Coupling ◽  
Protein Coupling ◽  
The Third ◽  
Third Intracellular Loop

scholarly journals The role of the third intracellular loop of the neutrophil N-formyl peptide receptor in G protein coupling

1993 ◽  
Vol 294 (2) ◽  
pp. 581-587 ◽  
Author(s):  
E R Prossnitz ◽  
O Quehenberger ◽  
C G Cochrane ◽  
R D Ye
Keyword(s):  
Amino Acids ◽  
G Protein ◽  
Formyl Peptide Receptor ◽  
Wild Type ◽  
Intracellular Loop ◽  
G Protein Coupling ◽  
Protein Coupling ◽  
The Third ◽  
Third Intracellular Loop ◽  
Formyl Peptide

The G-protein-coupled N-formyl peptide receptor (FPR) contains one of the smallest known third intracellular loops of this class of receptors, consisting of only 15 amino acids. To study the role of this region of the receptor in G protein coupling and signal transduction, we generated a deletion mutant (D3i) in which 10 amino acids of the loop were removed, as well as a series of site-directed mutants containing substitutions of the charged and polar amino acids of this loop. The D3i mutant, expressed at normal levels on the cell surface, displayed a KD for labelled N-formyl-Met-Leu-Phe ([3H]FMLP) of 165 nM. This value compares with a KD for the wild-type FPR of 1.0 nM, or 20 nM in the presence of guanosine 5′-[gamma-thio]triphosphate, which uncouples G proteins from the receptor. These results indicate that D3i contains significant structural defects, beyond the disruption of G protein coupling, that affect ligand binding properties. Ten site-directed mutants generated in the third intracellular loop (T226A, K227E, H229A, K230Q, K235Q, S236A, S236A/S237G, R238G, R241E and S244A) displayed KD values between 0.5 and 1.0 nM, with expression levels between 22% (K227E) and 111% (H229A) of that of wild type receptor. The capacity of the mutants for signal transductions was determined by measuring intracellular Ca2+ mobilization. Eight of the ten mutants displayed EC50 values for FMLP of between 0.07 and 0.9 nM, as compared with 0.12 nM for the wild-type receptor. The two mutants K227E and R238G had EC50 values of 2.7 and 2.9 nM respectively. The increase in EC50 could be accounted for partially by the low levels of receptor expression. All ten mutants gave maximum levels of Ca2+ mobilization similar to that produced by the wild-type FPR. These results contradict the conclusions reached with other G-protein-coupled receptors and indicate that the third intracellular loop of the FPR does not have a critical role in the functional coupling of G proteins.

scholarly journals Amino acid residues in third intracellular loop of melanocortin 1 receptor are involved in G-protein coupling

IUBMB Life ◽  
1998 ◽  
Vol 46 (5) ◽  
pp. 913-922
Author(s):  
Per-Anders Frändberg ◽  
Marina Doufexis ◽  
Supriya Kapas ◽  
Vijay Chhajlani
Keyword(s):  
Amino Acid ◽  
G Protein ◽  
Amino Acid Residues ◽  
Intracellular Loop ◽  
G Protein Coupling ◽  
Melanocortin 1 Receptor ◽  
Protein Coupling ◽  
Third Intracellular Loop

scholarly journals Intracellular Loop 2 Peptides of the Human 5HT1a Receptor are Differential Activators of Gi

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Brian Hall ◽  
Carley Squires ◽  
Keith K. Parker
Keyword(s):  
G Protein ◽  
Pharmacological Intervention ◽  
Intracellular Camp ◽  
Intracellular Loop ◽  
Peptide Mimics ◽  
G Protein Coupling ◽  
5Ht1a Receptor ◽  
Protein Coupling ◽  
Loop Region ◽  
Loop 2

Peptide mimics of intracellular loop 2 (ic2) of the human 5HT1a receptor have been studied with respect to their ability to inhibit agonist binding via interference with receptor-G-protein coupling. These peptides give shallow concentration-effect relationships. Additionally, these peptides have been studied with respect to their ability to trigger the signal transduction system of this Gi-coupled receptor. Two signaling parameters have been quantified: concentration of intracellular cAMP and changes in incorporation into the G protein of a stable analog of GTP. In both cases, peptide mimics near midloop of ic2 actually show agonist activity with efficacy falling off toward both loop termini near TM 3 and TM 4. Previous results have suggested that the loop region near the TM3/ic2 interface is primarily responsible for receptor-G-protein coupling, while the current result emphasizes the mid-ic2 loop region's ability to activate the G protein following initial coupling. A limited number of peptides from the receptor's TM5/ic3 loop vicinity were also studied regarding agonist inhibition and G-protein activation. These peptides provide additional evidence that the human 5HT1a receptor, TM5/ic3 loop region, is involved in both coupling and activation actions. Overall, these results provide further information about potential pharmacological intervention and drug development with respect to the human 5HT1a receptor/G-protein system. Finally, the structural evidence generated here provides testable models pending crystallization and X-ray analysis of the receptor.

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