Ligand-dependent differences in the regulation and internalization of the μ-opioid receptor (MOR) have been linked to the severity of adverse effects that limit opiate use in pain management. MOR activation by morphine or [D-Ala2,N-MePhe4, Gly-ol]enkephalin (DAMGO) causes differences in spatiotemporal signaling dependent on MOR distribution at the plasma membrane. Morphine stimulation of MOR activates a Gαi/o-Gβγ-protein kinase C (PKC) α phosphorylation pathway that limits MOR distribution and is associated with a sustained increase in cytosolic extracellular signal-regulated kinase (ERK) activity. In contrast, DAMGO causes a redistribution of the MOR at the plasma membrane (before receptor internalization) that facilitates transient activation of cytosolic and nuclear ERK. Here, we used proximity biotinylation proteomics to dissect the different protein-interaction networks that underlie the spatiotemporal signaling of morphine and DAMGO. We found that DAMGO, but not morphine, activates Ras-related C3 botulinum toxin substrate 1 (Rac1). Both Rac1 and nuclear ERK activity depended on the scaffolding proteins IQ motif-containing GTPase-activating protein-1 (IQGAP1) and Crk-like (CRKL) protein. In contrast, morphine increased the proximity of the MOR to desmosomal proteins, which form specialized and highly-ordered membrane domains. Knockdown of two desmosomal proteins, junction plakoglobin or desmocolin-1, switched the morphine spatiotemporal signaling profile to mimic that of DAMGO, resulting in a transient increase in nuclear ERK activity. The identification of the MOR-interaction networks that control differential spatiotemporal signaling reported here is an important step toward understanding how signal compartmentalization contributes to opioid-induced responses, including anti-nociception and the development of tolerance and dependence.