Activation of ERK by Wild Type and Mutant PAF Receptors

Laura de Marchena
Duke University

Platelet-activating factor (PAF) is an important phospholipid mediator that affects biological responses from the central nervous, respiratory, reproductive, and immune systems via the seven transmembrane domain PAF receptor (PAFR). Stimulation of the PAFR results in activation of a variety of signaling cascades, including MAP kinases. While this MAP kinase activation has traditionally been shown to require receptor coupling to heterotrimeric G proteins Gi and/or Gq, recent studies focusing on the angiotensin receptor have demonstrated a role for beta-arrestin 2 in the activation of MAP kinase extracellular signal-regulated kinase 1 and 2 (ERK 1/2) independent of G proteins. The aim of this study is to test if the PAFR also activates ERK in a beta-arrestin 2 dependent manner. To aid in the elucidation of the PAFR signaling capabilities, two versions of the PAFR were made with mutations that hinder G protein coupling (Y293A) or hinder both G protein coupling and internalization (D289A). The wild type and mutant receptors are being evaluated for ERK activation under several conditions to test the dependence of ERK signaling upon a particular variable. G protein dependence is being tested both for each type of G protein individually and for the effect of their combined loss on ERK activation. Gi dependence is being tested by inhibition with Pertussis Toxin, and Gq dependence by inhibition using the PKC inhibitor RO-31-8425. Finally, dependence on beta-arrestin 2 is being tested by depletion of cellular beta-arrestin 2 using RNA interference techniques.