Induced recruitment and activation of Fyn kinase to the GHS-R1a receptor by ghrelin.

<p>(<b>A</b>) Ghrelin activates Src-related kinases. Differentiated THP-1 cells were serum-deprived for 16 h and incubated with 1 µM Src-related kinase inhibitor PP2 or 10 nM PI3-K inhibitor LY-294002 for 30 min before incubation with 10 nM ghrelin for the indicated times. The lysates were then analyzed by immunoblot with an anti-phospho-specific to Src Tyr-418, the activation loop site, and loading was monitored with a β-actin antibody. (<b>B</b>) Ghrelin promotes Fyn activation in THP-1 macrophages. Cells were treated as in (A) and extracts were immunoprecipitated with an antibody against Fyn, and analyzed by immunoblot using anti-Src-pY418 and anti-Fyn antibodies. (<b>C</b>) Fyn is recruited to the GHS-R1a receptor and activated by ghrelin. THP-1 cells were treated similar as in (A) and immunoprecipitation was carried out with an antibody against GHS-R1a, and analyzed by immunoblot using anti-Src-pY418 and anti-Fyn antibodies. Samples were normalized with an anti-GHS-R1a antibody. (<b>D</b>) Effect of GHS-R1a inhibition on Fyn recruitment and activation. Serum deprived THP-1 cells were pretreated with 100 nM d-Lys³-GHRP-6 for 30 min, followed by incubation with 10 nM ghrelin for 30 min, and then analyzed as in (C).