THE ROLE OF THE 3RD INTRACELLULAR LOOP OF THE NEUTROPHIL N-FORMYL PEPTIDE RECEPTOR IN G-PROTEIN COUPLING

The G-protein-coupled N-formyl peptide receptor (FPR) contains one of the smallest known third intracellular loops of this class of receptor s, consisting of only 15 amino acids. To study the role of this region of the receptor in G protein coupling and signal transduction, we gen erated a deletion mutant (D3i) in which 10 amino acids of the loop wer e removed, as well as a series of site-directed mutants containing sub stitutions of the charged and polar amino acids of this loop. The D3i mutant, expressed at normal levels on the cell surface, displayed a K( D) for labelled N-formyl-Met-Leu-Phe ([H-3]FMLP) of 165 nM. This value compares with a K(D) 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 s ignificant structural defects, beyond the disruption of G protein coup ling, 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 K(D) va lues 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 intracell ular 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 o f 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 mutan ts gave maximum levels of Ca2+ mobilization similar to that produced b y the wild-type FPR. These results contradict the conclusions reached with other G-protein-coupled receptors and indicate that the third int racellular loop of the FPR does not have a critical role in the functi onal coupling of G proteins.