Barcoding of GPCR trafficking and signaling through the various trafficking roadmaps by compartmentalized signaling networks

Abstract

Proper signaling by G protein coupled receptors (GPCR) is dependent on the specific repertoire of transducing, enzymatic and regulatory kinases and phosphatases that shape its signaling output. Activation and signaling of the GPCR through its cognate G protein is impacted by G protein-coupled receptor kinase (GRK)-imprinted "barcodes" that recruit β-arrestins to regulate subsequent desensitization, biased signaling and endocytosis of the GPCR. The outcome of agonist-internalized GPCR in endosomes is also regulated by sequence motifs or "barcodes" within the GPCR that mediate its recycling to the plasma membrane or retention and eventual degradation as well as its subsequent signaling in endosomes. Given the vast number of diverse sequences in GPCR, several trafficking mechanisms for endosomal GPCR have been described. The majority of recycling GPCR, are sorted out of endosomes in a "sequence-dependent pathway" anchored around a type-1 PDZ-binding module found in their C-tails. For a subset of these GPCR, a second "barcode" imprinted onto specific GPCR serine/threonine residues by compartmentalized kinase networks was required for their efficient recycling through the "sequence-dependent pathway". Mutating the serine/threonine residues involved, produced dramatic effects on GPCR trafficking, indicating that they played a major role in setting the trafficking itinerary of these GPCR. While endosomal SNX27, retromer/WASH complexes and actin were required for efficient sorting and budding of all these GPCR, additional proteins were required for GPCR sorting via the second "barcode". Here we will review recent developments in GPCR trafficking in general and the human β₁-adrenergic receptor in particular across the various trafficking roadmaps. In addition, we will discuss the role of GPCR trafficking in regulating endosomal GPCR signaling, which promote biochemical and physiological effects that are distinct from those generated by the GPCR signal transduction pathway in membranes.

Keywords: Endosomes; GPCR; GRK; PKA; SNX27; Signaling; Trafficking.

Fig. 1. A, Structure of A-kinase anchoring protein79/150 [AKAP5] and B, SAP97

Fig. 2. Organization of the ß₁-adrenergic receptosome around the type-1 PBM in the carboxy-tail of the ß₁-AR

The figure illustrates bipartite binding of SAP97 simultaneously to the ß₁-AR PBM (via its second PDZ) and to AKAP5 (via the I3 domain of SAP97). The figure indicates that PDE4D8 was bound to SAP97 as well. Activation of the ß₁-AR signaling pathway in the membranes increases cyclic AMP, which activates PKA. Spatiotemporal activity of cyclic AMP is contained through its degradation by receptosomal PDE4D8. Activated PKA in turn, faithfully phosphorylates the ß₁-AR on the serine at position 312 to imprint a recycling signal onto the ß₁-AR prior to its sequestration.

Fig. 3. Schematic illustrating the role of endosomal trafficking proteins in regulating the GPCR recycling by the one- or two barcode paradigms

A, Organization of SNX27 into its three major protein binding domains. B. Structure of retromer and WASH complexes that are involved in endosomal trafficking. The retromer is composed of a cargo selection trimer (CST composed of Vps 35, 29 and 26) and BAR dimers. The retromer is recruited to endosomes via PtdIns(3)P-binders such as the GTPase Rab7a, and sorting nexin-3 (SNX-3). CST interacts with the WASH complex via the C-tail of FAM21 that also binds to SNX27 and FKBP15 to mediate endosome to plasma membrane trafficking. C, The SNX27 interactome. PBM-containing cargo binds to the PDZ of SNX27; while the PX and FERM domains of SNX27 are involved in recruiting SNX27 to endosomes and to the WASH complex respectively. In addition, a domain in SNX27 between 66-76 binds to Vps26 of the retromer. Adapted from [120]. D. Domain organization of FKBP15. E. Schematic of GPCR trafficking in endosomes. Activation of membranous ß₁-AR promotes their internalization into early endosomes (red arrows). The type-1 PBM of the ß₁-AR (blue) binds to the PDZ of SNX27 (green). SNX27 directs PBM containing cargoes into tubules that utilize actin/retromer/WASH complexes (termed ASRT) for budding out of early endosomes. This mode of trafficking is mediated by the PBM “barcode” and is sufficient to guide most GPCR out of early endosomes. However, tubulation and budding of WT ß₁-AR out of endosomes required a second “barcode”, which was phospho-Ser³¹². This reversible “barcode” was imprinted by the ß₁-adrenergic receptosome and is recognized by endosomal sorting proteins, such as FKBP15. Upon verifying its two barcodes, the ß₁-AR is sorted from early endosomes into rab11 containing recycling endosomes and then re-inserted into the plasma membrane (blue arrows). Inhibition of PKA or inactivation of either barcode leads to retention of these cargoes within the body of early endomes (orange rectangle), which eventually mature into late endosomes/lysosomes. ß₁-AR constructs with recycling nonPDZ sequences (black cube) or with a Ser³¹² to aspartic acid mutation are sorted out of early endosomes in a retromer/WASH independent pathway (light green rectangle).