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Mariggió, Stefania Pasqua
(2001).
DOI: https://doi.org/10.21954/ou.ro.0000fce2
Abstract
Biological systems consistently diminish their responses to persistent or stable stimuli, in a process termed desensitisation or adaptation. This study focuses on the process called “homologous desensitisation” that indicates the rapid and reversible loss of receptor responsiveness that occurs upon exposure to agonists (Premont, 1995; Palczewski, 1997). Key molecular determinants of this process are G protein-coupled receptor kinases (GRKs) and arrestins (Chuang, 1996a). Both GRKs and arrestins are expressed at high level in peripheral blood leukocytes (PBL) and they are potentially modulators of *chemoattractant receptor-mediated immune responses (Parruti, 1993; Craft, 1995; Pitcher, 1998a). We studied human Platelet-Activating Factor receptor (hPAFR) signalling and its modulation. We showed that in a heterologous expression system recombinant hPAFR stimulated inositol phosphates production and intracellular cAMP accumulation, through the coupling with Gq/ 11 and Gs proteins, respectively. The Gs-coupling was a totally unpredicted finding. We also showed that PAF could stimulate intracellular cAMP accumulation in lymphocytes, suggesting that PAFR-coupling with adenylyl cyclase may be important also for endogenously expressed receptors. With the co-transfection approach we defined which GRKs were able to modulate hPAFR signalling and the mechanisms involved in this modulation. We showed that receptor pathways mediated by Gs-coupling were regulated through a strictly phosphorylation-dependent process, while Gq-mediated signalling could be modulated in a phosphorylation-independent way. We also identified in the N-terminal portion of GRK2 (N-ter) the domain involved in the down regulation of Gq-mediated signalling. This inhibition is likely through direct binding, as demonstrated by specific and selective interaction between N-ter and activated Gaq in vitro binding assays and co-immunoprecipitation studies. We also show that GRK2 binding to Gaq can be regulated by c-Src activity. In particular, GRK2 tyrosine-phosphorylation, mediated by c-Src, increases GRK2 affinity for Gaq, reinforcing the desensitisation pathway.