Monitoring the cellular trafficking of prostate-specific membrane antigen via bio-labeling with non-canonical amino acids.

Prostate-Specific Membrane Antigen (PSMA) is a well-established target for prostate cancer therapeutics, most of which rely on receptor internalization for efficacy. However, how different ligands modulate PSMA internalization and plasma membrane residence time remains poorly understood. This study aims to characterize the intracellular trafficking dynamics of PSMA—both in the absence and presence of anti-PSMA antibodies—with a focus on its membrane retention kinetics. To achieve high spatiotemporal resolution, we employ fluorescence microscopy combined with site-specific incorporation of non-canonical amino acids (ncAAs) bearing fluorescent handles. This labeling strategy was adopted because conventional eGFP fusion disrupted correct PSMA trafficking to the ER, underscoring the need for a minimally perturbing approach. We selected the amber codon suppression system (Schultz et al.) to incorporate p-azide-phenylalanine (pAzF) during PSMA biosynthesis. Here, we present the detailed construction of this system in HeLa cells: an engineered orthogonal tRNA/tRNA synthetase pair specific for pAzF is co-expressed with PSMA containing an amber codon at the desired position, allowing pAzF incorporation by competing with termination factors at the UAG codon. Following pAzF derivatization with Cy3 via click chemistry, we are currently evaluating, by time-lapse fluorescence microscopy, whether PSMA-targeting nanobodies with different affinities modulate receptor residency at the plasma membrane. Additionally, to enable precise kinetic control, we will implement the RUSH (Retention Using Selective Hooks) system, permitting synchronized biotin-dependent release of a defined PSMA pool from the ER and real-time monitoring of a synchronized protein population. Our findings may inform the rational design of next-generation PSMA-targeted therapeutics with optimized pharmacokinetics and enhanced efficacy for prostate cancer treatment.