Scribble scrambles parathyroid hormone receptor interactions to regulate phosphate and vitamin D homeostasis.

G protein-coupled receptors, including PTHR, are pivotal for controlling metabolic processes ranging from serum phosphate and vitamin D levels to glucose uptake, and cytoplasmic interactors may modulate their signaling, trafficking, and function. We now show that direct interaction with Scribble, a cell polarity-regulating adaptor protein, modulates PTHR activity. Scribble is a crucial regulator for establishing and developing tissue architecture, and its dysregulation is involved in various disease conditions, including tumor expansion and viral infections. Scribble co-localizes with PTHR at basal and lateral surfaces in polarized cells. Using X-ray crystallography, we show that colocalization is mediated by engaging a short sequence motif at the PTHR C-terminus using Scribble PDZ1 and PDZ3 domain, with binding affinities of 31.7 and 13.4 µM, respectively. Since PTHR controls metabolic functions by actions on renal proximal tubules, we engineered mice to selectively knockout Scribble in proximal tubules. The loss of Scribble impacted serum phosphate and vitamin D levels and caused significant plasma phosphate elevation and increased aggregate vitamin D3 levels, whereas blood glucose levels remained unchanged. Collectively these results identify Scribble as a vital regulator of PTHR-mediated signaling and function. Our findings reveal an unexpected link between renal metabolism and cell polarity signaling.

Structural analysis of human papillomavirus E6 interactions with Scribble PDZ domains.

The cell polarity regulator Scribble has been shown to be a critical regulator of the establishment and development of tissue architecture, and its dysregulation promotes or suppresses tumour development in a context-dependent manner. Scribble activity is subverted by numerous viruses. This includes human papillomaviruses (HPVs), who target Scribble via the E6 protein. Binding of E6 from high-risk HPV strains to Scribble via a C-terminal PDZ-binding motif leads to Scribble degradation in vivo. However, the precise molecular basis for Scribble-E6 interactions remains to be defined. We now show that Scribble PDZ1 and PDZ3 are the major interactors of HPV E6 from multiple high-risk strains, with each E6 protein displaying a unique interaction profile. We then determined crystal structures of Scribble PDZ1 and PDZ3 domains in complex with the PDZ-binding motif (PBM) motifs of E6 from HPV strains 16, 18 and 66. Our findings reveal distinct interaction patterns for each E6 PBM motif from a given HPV strain, suggesting that a complex molecular interplay exists that underpins the overt Scribble-HPV E6 interaction and controls E6 carcinogenic potential.

Crystallographic Studies of PDZ Domain-Peptide Interactions of the Scribble Polarity Module.

The determination of high-resolution crystal structures of cell polarity regulatory proteins bound to their functional interactors has proven to be invaluable for deciphering the underlying molecular mechanisms. Here we describe methods to identify suitable complexes of cell polarity protein domains bound to interacting ligands with subsequent preparation of such complexes for X-ray crystallographic analysis.

Structural analysis of phosphorylation-associated interactions of human MCC with Scribble PDZ domains.

Scribble is a crucial adaptor protein that plays a pivotal role during establishment and control of cell polarity, impacting many physiological processes ranging from cell migration to immunity and organization of tissue architecture. Scribble harbours a leucine-rich repeat domain and four PDZ domains that mediate most of Scribble's interactions with other proteins. It has become increasingly clear that post-translational modifications substantially impact Scribble-ligand interactions, with phosphorylation being a major modulator of binding to Scribble. To better understand how Scribble PDZ domains direct cell polarity signalling and how phosphorylation impacts this process, we investigated human Scribble interactions with MCC (Mutated in Colorectal Cancer). We systematically evaluated the ability of all four individual Scribble PDZ domains to bind the PDZ-binding motif (PBM) of MCC as well as MCC phosphorylated at the -1 Ser position. We show that Scribble PDZ1 and PDZ3 are the major interactors with MCC, and that modifications to Ser at the -1 position in the MCC PBM only has a minor effect on binding to Scribble PDZ domains. We then examined the structural basis for these observations by determining the crystal structures of Scribble PDZ1 domain bound to both the unphosphorylated MCC PBM as well as phosphorylated MCC. Our structures indicated that phospho-Ser at the -1 position in MCC is not involved in major contacts with Scribble PDZ1, and in conjunction with our affinity measurements suggest that the impact of phosphorylation at the -1 position of MCC must extend beyond a simple modulation of the affinity for Scribble PDZ domains.

Drosophila melanogaster Guk-holder interacts with the Scribbled PDZ1 domain and regulates epithelial development with Scribbled and Discs Large.

Epithelial cell polarity is controlled by components of the Scribble polarity module, and its regulation is critical for tissue architecture and cell proliferation and migration. In Drosophila melanogaster, the adaptor protein Guk-holder (Gukh) binds to the Scribbled (Scrib) and Discs Large (Dlg) components of the Scribble polarity module and plays an important role in the formation of neuromuscular junctions. However, Gukh's role in epithelial tissue formation and the molecular basis for the Scrib-Gukh interaction remain to be defined. We now show using isothermal titration calorimetry that the Scrib PDZ1 domain is the major site for an interaction with Gukh. Furthermore, we defined the structural basis of this interaction by determining the crystal structure of the Scrib PDZ1-Gukh complex. The C-terminal PDZ-binding motif of Gukh is located in the canonical ligand-binding groove of Scrib PDZ1 and utilizes an unusually extensive network of hydrogen bonds and ionic interactions to enable binding to PDZ1 with high affinity. We next examined the role of Gukh along with those of Scrib and Dlg in Drosophila epithelial tissues and found that Gukh is expressed in larval-wing and eye-epithelial tissues and co-localizes with Scrib and Dlg at the apical cell cortex. Importantly, we show that Gukh functions with Scrib and Dlg in the development of Drosophila epithelial tissues, with depletion of Gukh enhancing the eye- and wing-tissue defects caused by Scrib or Dlg depletion. Overall, our findings reveal that Scrib's PDZ1 domain functions in the interaction with Gukh and that the Scrib-Gukh interaction has a key role in epithelial tissue development in Drosophila.