Calcium-Dependent Association of Calmodulin with the C-Terminal Domain of the Tetraspanin Protein Peripherin/rds

Abstract

Peripherin/rds (p/rds), an integral membrane protein from the transmembrane 4 (TMF4) superfamily, possesses a multi-functional C-terminal domain that plays crucial roles in rod outer segment (ROS) disk renewal and structure. Here, we report that the calcium binding protein calmodulin (CaM) binds to the C-terminal domain of p/rds. Fluorescence spectroscopy reveals Ca 2+ -dependent association of CaM with a polypeptide corresponding to the C-terminal domain of p/rds. The fluorescence anisotropy of the polypeptide upon CaM titration yields a dissociation constant (K D ) of 320 ± 150 nM. The results of the fluorescence experiments were confirmed by GST-pull down analyses in which a GST-p/rds C-terminal domain fusion protein was shown to pull down CaM in a calcium-dependent manner. Moreover, molecular modeling and sequence predictions suggest that the CaM binding domain resides in a p/rds functional hot spot, between residues E314 and G329. Predictions were confirmed by peptide competition studies and a GSTp/rds C-terminal domain construct in which the putative Ca 2+ /CaM binding site was scrambled. This GST-polypeptide did not associate with Ca 2+ /CaM. This putative calmodulin domain is highly conserved between human, mouse, rat, and bovine p/rds. Finally, the binding of Ca 2+ /CaM inhibited fusion between ROS disk and ROS plasma membranes as well as p/rds C-terminaldomain-induced fusion in model membrane studies. These results offer a new mechanism for the modulation of p/rds function.Photoreceptor peripherin/rds (p/rds 1 ) and the non-glycosylated homologue ROM-1 belong to a growing and increasingly complex family of proteins, the tetraspanins (also known as the TMF4 (transmembrane 4 superfamily)) (1, 2). P/rds shares 41% sequence homology

PRPH2/RDS and ROM-1: Historical context, current views and future considerations

“…When MREG was added to PRPH2/RDS membrane fusion assays, the rate of membrane fusion was significantly reduced, suggesting that MREG acts as an inhibitor of PRPH2/RDS’ fusogenic activity (Boesze-Battaglia et al, 2007a). The C-terminus can also associate with the calcium binding protein calmodulin in a Ca 2+ dependent manner (Edrington et al, 2007b). Similarly to MREG, this binding seems to inhibit the fusion capacity of the PRPH2/RDS protein and binds to a similar region of the protein (Edrington et al, 2007b).…”

“…The C-terminus can also associate with the calcium binding protein calmodulin in a Ca 2+ dependent manner (Edrington et al, 2007b). Similarly to MREG, this binding seems to inhibit the fusion capacity of the PRPH2/RDS protein and binds to a similar region of the protein (Edrington et al, 2007b). The mechanisms of PRPH2/RDS fusogenic activity and its role in OS biogenesis is not fully elucidated, it is clear that PRPH2/RDS fusogenic activity involves the PRPH2/RDS C-terminus and can be regulated by various binding partners.…”

Structural and functional relationships between photoreceptor tetraspanins and other superfamily members

“…CaM binds RDS in a calcium-dependent manner [49]. The putative CaM binding site is predicted to be between residues E314 and G329, roughly corresponding to the membrane fusion domain of the RDS C-terminus.…”

“…The putative CaM binding site is predicted to be between residues E314 and G329, roughly corresponding to the membrane fusion domain of the RDS C-terminus. It has been proposed that this binding plays a role in the apical shedding of the rod OS by regulating the fusogenic properties of the RDS C-terminus [49]. MREG and CaM binding data suggest that RDS-mediated membrane fusion is differently regulated based on subcellular localization of the interacting partners and cellular conditions.…”

Peripherin/rds co-distributes with putative binding partners in basal rod outer segment disks

“…As the peripherin/rds protein traffics up the transition zone to aid in the formation of new disks it has numerous binding partners, including a cargo sorting protein melanoregulin (MREG) as well as Calcium/Calmodulin (Ca/CaM) (Boesze-Battaglia, 2007; Edrington, 2007a). These functional and binding regions of peripherin/rds are illustrated in Fig.1.…”