Sparsely Phosphorylable Residues, Tuned by Agonist Chemotype, Shapes GPCR Signaling

GPCR signaling diversity is often attributed to an intracellular phosphorylation “barcode,” in which ligand-dependent modification of multiple receptor phosphoresidues encodes selective engagement of downstream transducers. However, whether such an extensive phosphorylation of the receptor is universally required remains unclear. Here, we show that the melanocortin-4 receptor (MC4R), a central regulator of energy homeostasis, achieves remarkably diverse and pathway-selective signaling through a sparse set of strategically positioned regulatory phosphoresidues. Systematic study of these phosphoresidues within helix 8 and the C-terminal tail revealed distinct, spatially segregated contributions to receptor function. Substitution of helix 8 residues S306 disrupted β-arrestin2 recruitment, early endosomal trafficking, and ERK1/2 activation, while visibly altering Gαs–cAMP signaling in all the receptor variants. In contrast, distal C-terminal substitutions had little effect on β-arrestin2 coupling and trafficking, revealing that these regions are not functionally redundant. Surprisingly, signaling through Gα _q/11 -and Gα _12/13 -linked pathways remained largely constrained across receptor variants, further demonstrating pathway-specific control by these phosphoresidues. Notably, ligand chemotype reshaped the functional consequences of these substitutions: setmelanotide selectively restored or enhanced signaling through pathways that were compromised by specific receptor mutations. Together, our findings argue that GPCR signaling diversity does not necessarily require an extensive phosphorylation barcode. Instead, MC4R uses a minimal number of spatially organized phosphoresidue that can differentially control transducer engagement and downstream signaling, while ligand chemistry determines how this regulatory landscape is accessed.