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†These authors contributed equally to the work.Activated human neuropeptide Y Y 1 receptors rapidly desensitize and internalize through clathrin-coated pits and recycle from early and recycling endosomes, unlike Y 2 receptors that neither internalize nor desensitize. To identify motifs implicated in Y 1 receptor desensitization and trafficking, mutants with varying C-terminal truncations or a substituted Y 2 C-terminus were constructed. Point mutations of key putative residues were made in athat we have identified and in the second intracellular i2 loop. Receptors were analyzed by functional assays, spectrofluorimetric measurements on living cells, flow cytometry, confocal imaging and bioluminescence resonance energy transfer assays for b-arrestin activation and adaptor protein (AP-2) complex recruitment. Inhibitory GTP-binding protein-dependent signaling of Y 1 receptors to adenylyl cyclase and desensitization was unaffected by C-terminal truncations or mutations, while C-terminal deletion mutants of 42 and 61 amino acids no longer internalized. Substitutions of Thr357, Asp358, Ser360 and Thr362 by Ala in the C-terminus abolished both internalization and b-arrestin activation but not desensitization. A Pro145 substitution by His in an i2 consensus motif reported to mediate phosphorylation-independent recruitment of b-arrestins affected neither desensitization, internalization or recycling kinetics of activated Y 1 receptors nor b-arrestin activation. Interestingly, combining Pro145 substitution by His and C-terminal substitutions significantly attenuates Y 1 desensitization. In the Y 2 receptor, replacement of His155 with Pro at this position in the i2 loop motif promotes agonist-mediated desensitization, b-arrestin activation, internalization and recycling. Overall, our results indicate that b-arrestin-mediated desensitization and internalization of Y 1 and Y 2 receptors are differentially regulated by the C-terminal motif and the i2 loop consensus motif.

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