PT  - JOURNAL ARTICLE
AU  - Michael Schumann
AU  - Tomoo Nakagawa
AU  - Samuel A. Mantey
AU  - Kenji Tokita
AU  - David J. Venzon
AU  - Simon J. Hocart
AU  - Richard V. Benya
AU  - Robert T. Jensen
TI  - Importance of Amino Acids of the Central Portion of the Second Intracellular Loop of the Gastrin-Releasing Peptide Receptor for Phospholipase C Activation, Internalization, and Chronic Down-Regulation
AID  - 10.1124/jpet.103.055087
DP  - 2003 Nov 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 597--607
VI  - 307
IP  - 2
4099  - http://jpet.aspetjournals.org/content/307/2/597.short
4100  - http://jpet.aspetjournals.org/content/307/2/597.full
SO  - J Pharmacol Exp Ther2003 Nov 01; 307
AB  - Little is known about the function of the central portion of the second intracellular loop (i2 loop) of peptide receptors in activation of downstream pathways and receptor modulatory processes such as receptor internalization or chronic down-regulation (DR). Recent data suggest a role for i2 loop hydrophobic amino acids in these processes. We used site-directed mutagenesis to address these issues with the gastrin-releasing peptide receptor (GRP-R). Each i2 loop residue from 142 to 148 was mutated and the receptors were expressed in Balb 3T3 cells. Two mutants showed a minimal (<2-fold) decrease in affinity. Five mutants showed decreased efficacy for activating phospholipase C (PLC). Two double mutants (IM143.147AA and VM144.147AA) showed a minimal decrease in affinity but had a decreased ability to fully activate PLC. Only the IM double mutation had decreased maximal internalization, whereas the R145A single mutant showed an increase, suggesting a tonic inhibitory role for Arg-145 in internalization. Three single and both double mutants showed decreases in receptor DR. There was a weak correlation between the extent of GRP-R internalization and the maximal PLC activation, whereas changes in the maximal PLC activation were significantly (p = 0.008) coupled to receptor DR. This study shows that amino acids of the i2 loop of the GRP-R are important in activation of PLC, internalization and down-regulation, but not for affinity. Our results support the proposal that internalization and chronic down-regulation have differing dependence on PLC and are largely independent processes, because some mutants showed no changes in internalization, but significant alterations in down-regulation. The American Society for Pharmacology and Experimental Therapeutics