SHANK3 conformation regulates direct actin binding and crosstalk with Rap1 signaling

Abstract

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology, and specific biological functions of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators, and small guanosine triphosphatases (GTPases). SHANK3 is a multifunctional scaffold protein, interacting with several actin-binding proteins and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its Shank/ProSAP N-terminal (SPN) domain. Here, we demonstrate that, in addition to scaffolding actin regulators and actin-binding proteins, SHANK3 interacts directly with actin through its SPN domain. Molecular simulations and targeted mutagenesis of the SPN-ankyrin repeat region (ARR) interface reveal that actin binding is inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3, and mutation of SHANK3, resulting in reduced actin binding, augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for cell morphology and integrin activity in cancer cells. In addition, SHANK3-actin interaction regulates dendritic spine morphology in neurons and autism-linked phenotypes in vivo.

Mutations affecting the N-terminal domains of SHANK3 point to different pathomechanisms in neurodevelopmental disorders

“…The change of Asn52 to Arg in the SPN domain has been designed based on the 3D-structure, with the intention to weaken the affinity of the SPN to the Ank domain. Indeed we have shown before that it truly opens up the SPN − Ank interaction 32 . Upon coexpression of WT and mutant forms of the complete N-terminus with the GFP-tagged SPN domain, only the N52R positive control induced a significant increase in binding of the SPN domain to the N-terminus (Fig.…”

“…7 a). We analysed the P141A mutation, together with two controls for which we were confident that they would disrupt the intramolecular contact between SPN and Ank domains: N52R (see above, and 32 ) and L68P, a patient mutation for which we have shown that it disrupts the folding of the SPN domain 18 , 23 , 33 . We also included, in a separate experiment, a mutant which is not involved in the interface between SPN and Ank: R12C.…”

Scaffold proteins as dynamic integrators of biological processes

“…Salomaa et al . ( 48 ) demonstrated a role for SHANK3 in the cytoskeletal organization of the PSD by modulation of its active (open) and inactive (closed) conformations ( Fig. 4 A ).…”

“…SHANK3 appears to act as a master regulator of the PSD by organizing cytoskeletal elements to support synaptic signaling. SHANK3 has several protein-binding domains including, among others, an N-terminal SPN domain that binds RAP1 and actin during integrin signaling, an SH3 domain that binds to AMPA receptor (AMPAR) complexes, and an ANK domain that binds cytoskeletal elements such as α-fodrin ( 48 , 49 , 50 , 51 ). Emerging evidence indicates that SHANK3 regulates the PSD, at least in part, through conformational changes that switch SHANK3 between active and inactive states based on its bound ligand ( 48 , 52 ).…”

“…SHANK3 has several protein-binding domains including, among others, an N-terminal SPN domain that binds RAP1 and actin during integrin signaling, an SH3 domain that binds to AMPA receptor (AMPAR) complexes, and an ANK domain that binds cytoskeletal elements such as α-fodrin ( 48 , 49 , 50 , 51 ). Emerging evidence indicates that SHANK3 regulates the PSD, at least in part, through conformational changes that switch SHANK3 between active and inactive states based on its bound ligand ( 48 , 52 ). Of note, loss-of-function mutations in SHANK3 are linked to neurodevelopmental disorders like autism spectrum disorder ( 49 ).…”