Abstract
The β1-adrenergic receptor (β1-AR) is a target for treatment of major cardiovascular diseases, such as heart failure and hypertension. Recycling of agonist-internalized β1-AR is dependent on type I PSD-95/DLG/ZO1 (PDZ) in the C-tail of the β1-AR and on protein kinase A (PKA) activity (Gardner, L. A., Naren, A. P., and Bahouth, S. W. (2007) J. Biol. Chem. 282, 5085-5099). We explored the effects of point mutations in the PDZ and in the activity of PKA on recycling of the β1-AR and its binding to the PDZ-binding protein SAP97. These studies indicated that β1-AR recycling was inhibited by PKA inhibitors and by mutations in the PDZ that interfered with SAP97 binding. The trafficking effects of short sequences differing in PDZ and SAP97 binding were examined using chimeric mutant β1-AR. β1-AR chimera containing the type I PDZ of the β2-adrenergic receptor that does not bind to SAP97 failed to recycle except when serine 312 was mutated to aspartic acid. β1-AR chimera with type I PDZ sequences from the C-tails of aquaporin-2 or GluR1 recycled in a SAP97- and PKA-dependent manner. Non-PDZ β1-AR chimera derived from μ-opioid, dopamine 1, or GluR2 receptors promoted rapid recycling of chimeric β1-AR in a SAP97- and PKA-independent manner. Moreover, the nature of the residue at position -3 in the PDZ regulated whether the β1-AR was internalized alone or in complex with SAP97. These results indicate that divergent pathways were involved in trafficking the β1-AR and provide a roadmap for its trafficking via type I PDZs versus non-PDZs.
Highlights
The 1-adrenergic receptor (1-AR) is a target for treatment of major cardiovascular diseases, such as heart failure and hypertension
The trafficking effects of short sequences differing in PDZ and SAP97 binding were examined using chimeric mutant 1-AR. 1-AR chimera containing the type I PDZ of the 2-adrenergic receptor that does not bind to SAP97 failed to recycle except when serine 312 was mutated to aspartic acid. 1-AR chimera with type I PDZ sequences from the C-tails of aquaporin-2 or GluR1 recycled in a SAP97- and protein kinase A (PKA)-dependent manner
NUMBER 4 centrations of their respective agonists (1, 2). This process is initiated by coordinated activities of G protein receptor kinase (GRK) and -arrestin to promote the internalization of the agonist-activated G protein-coupled receptors (GPCRs) (3, 4)
Summary
CDNA Constructs, siRNA, and Reagents—Modifications in the 1-AR were generated by PCR to introduce a FLAG sequence (DYKDDDDK) upstream of the N-terminal region and the desired mutation in the C terminus of the 1-AR. siRNA sequences to human SAP97, 5Ј-GATATCCAGGAACATAAAT-3Ј, or its control 5Ј-ccataatacaaGgtataa-3Ј were cloned in the pcDNA 6.2-GW/EmGFP-miR vector (Invitrogen). Cy3labeled cells were washed in HBSS and incubated in culture medium containing 0.1 mM ascorbic acid. Recycling of internal GPCR was initiated by culturing the cells with a 100 M concentration of the -antagonist alprenolol at 37 °C for 15, 30, or 60 min, followed by fixing the slides in 4% paraformaldehyde and 4% sucrose (pH 7.4). At the completion of recycling (i.e. the 60-min slide), the slide was exposed to a second acid wash to strip the Cy3-labeled antibody from the externalized receptor population and fixed. After 24 –36 h, the cells were labeled with Cy3-conjugated anti-FLAG IgG for 60 min at 10 °C. HEK-293 cells stably expressing equivalent amounts of the indicated FLAG-tagged -AR (between 0.9 and 1.3 pmol of 1-AR/mg protein) were cultured.
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call