Autoinhibition of the ligand-binding site of GGA1/3 VHS domains by an internal acidic cluster-dileucine motif

Abstract

The GGAs (Golgi-localizing, ␥-adaptin ear homology domain, ARFbinding proteins) are a family of proteins implicated in protein trafficking from the Golgi to endosomes͞lysosomes. These proteins have modular structures with an N-terminal VHS (VPS-27, Hrs, and STAM) domain followed by a GAT (GGA and TOM1) domain, a connecting hinge segment, and a C-terminal GAE (␥-adaptin ear) domain. Isolated VHS domains have been shown to bind specifically to acidic cluster (AC)-dileucine motifs present in the cytoplasmic tails of the mannose 6-phosphate receptors. Here we report that full-length cytoplasmic GGA1 and GGA3 but not GGA2 bind the cation-independent mannose 6-phosphate receptor very poorly because of autoinhibition. This inhibition is caused by the binding of an AC-LL sequence present in the hinge segment to the ligand-binding site in the VHS domain. The inhibition depends on the phosphorylation of a serine located three residues upstream of the AC-LL motif. The serine is phosphorylated by casein kinase 2 in in vitro assays. Substitution of the GGA1 inhibitory sequence into the analogous location in GGA2, which lacks the AC-LL motif, results in autoinhibition of the latter protein. These data indicate that the activity of GGA1 and GGA3 is regulated by cycles of phosphorylation͞dephosphorylation.

Functional characterization of protein-sorting machineries at the trans-Golgi network in Drosophila melanogaster

“…In this model, GGA is displaced by AP-1 through a phosphorylation statedependent structural change. Internal ACLL motifs in the hinge region of human GGA1 and GGA3 are thought to mediate this structural change (Doray et al, 2002c). Through the characterization of dGGA and Drosophila AP-1 (dAP-1) in the current study (Fig.…”

The Structure and Function of GGAs, the Traffic Controllers at the TGN Sorting Crossroads

“…Later, the GGA-VHS domains were shown to be able to interact with the ACLL sequence in b-secretase (BACE), which is responsible for the b-site cleavage of the amyloid precursor protein to yield the Ab peptide, and that in sorLA, which is homologous to sortilin (He et al, 2002;Jacobsen et al, 2002). Furthermore, it is also noteworthy that the VHS domains are able to interact with potential ACLL sequences that are found in the hinge regions of GGA1 and GGA3 but not in GGA2 (Doray et al, 2002a) (see below). Despite its binding to cargo proteins found in the TGN, the VHS domain appears not to be the major determinant of TGN association of GGAs, because the domain alone expressed in cells is predominantly cytosolic and because GGA3S, which is defective in ACLL binding, shows TGN localization.…”

“…2). Phosphorylation of a serine residue at position -3 results in autoinhibition of the VHS domain from interacting with the ACLL sequences of cargo proteins (Doray et al, 2002a). Based on the VHSpeptide complex structures, however, the serine residue is predicted to be placed out of the conventional ACLL binding site, suggesting that the phosphorylated serine is recognized by another part of the VHS domain.…”

“…2). It has been proposed that once the serine residue is phosphorylated, an autoinhibitory intramolecular interaction between the VHS domain and the phosphorylated ACLL sequence thereby occurs, rendering GGAs incapable of interacting with the ACLL sequences of cargo proteins (Doray et al, 2002a;Ghosh and Kornfeld, 2003).…”

Structure of the GAT domain of human GGA1: A syntaxin amino-terminal domain fold in an endosomal trafficking adaptor

“…The hinge region contains a clathrin-binding sequence and is responsible for clathrin association (8,11,13,14). The hinge region of GGA1 and GGA3 also contains an autoinhibitory sequence that, when phosphorylated by casein kinase II, blocks receptor binding to the VHS domain (15). The GAE domain is responsible for binding accessory proteins, including Rabaptin-5 and ␥-synergin (2,5,8,16).…”