Protein kinase A and G protein-coupled receptor kinase phosphorylation mediates β-1 adrenergic receptor endocytosis through different pathways

Antonio Rapacciuolo, Shayela Suvarna, Liza Barki-Harrington, Louis M. Luttrell, Mei Cong, Robert J. Lefkowitz, Howard A. Rockman

Research output: Contribution to journalArticlepeer-review

Abstract

Agonist-induced phosphorylation of β-adrenergic receptors (βARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization followed by internalization. Whether protein kinase A (PKA)-mediated phosphorylation of βARs, particularly the β1AR subtype, can also trigger internalization is currently not known. To test this, we cloned the mouse wild type β1AR (WTβ1AR) and created 3 mutants lacking, respectively: the putative PKA phosphorylation sites (PKA-β1AR), the putative GRK phosphorylation sites (GRK-β1AR), and both sets of phosphorylation sites (PKA-/ GRK-β1AR). Following agonist stimulation, both PKA-β1AR and GRK -β1AR mutants showed comparable increases in phosphorylation and desensitization. Saturating concentrations of agonist induced only 50% internalization of either mutant compared with wild type, suggesting that both PKA and GRK phosphorylation of the receptor contributed to receptor sequestration in an additive manner. Moreover, in contrast to the WTβ1AR and PKA-β1AR, sequestration of the GRK-β1AR and PKA-/GRK -β1AR was independent of β-arrestin recruitment. Importantly, clathrin inhibitors abolished agonist-dependent internalization for both the WTβ1AR and PKA-β1AR, whereas caveolae inhibitors prevented internalization only of the GRK -β1AR mutant. Taken together, these data demonstrate that: 1) PKA-mediated phosphorylation can trigger agonist-induced internalization of the β1AR and 2) the pathway selected for β1AR internalization is primarily determined by the kinase that phosphorylates the receptor, i.e. PKA-mediated phosphorylation directs internalization via a caveolae pathway, whereas GRK-mediated phosphorylation directs it through clathrin-coated pits.

Original languageEnglish
Pages (from-to)35403-35411
Number of pages9
JournalJournal of Biological Chemistry
Volume278
Issue number37
DOIs
StatePublished - 12 Sep 2003
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry
  • Cell Biology

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