The receptors for the neuropeptide calcitonin (CT) gene-related peptide (CGRP) and the multifunctional peptide hormone adrenomedullin (AM) are calcitonin-like receptor (CLR)/receptor-activity-modifying protein (RAMP) 1 and CLR/RAMP2 heterodimers, respectively. Here, the amino acid sequence TRNKIMT, corresponding to the residues 14-20 of the N terminus of the mouse (m) CLR, was found to be required for a functional mCLR/RAMP2 AM receptor. The deletion of amino acids 14-20 (Δ14-20) or their substitution by alanine (14-20A) did not affect the heterodimerization of the mCLR with mRAMP1 or mRAMP2, and the levels of expression at the surface of transiently transfected COS-7 cells were not altered. In mRAMP1/mCLR- or mRAMP1/mCLR-(Δ14-20)-expressing cells CGRP stimulated cAMP formation with EC50 values of 0.12 ± 0. 01 and 1.5 ± 0.4 nM, respectively. In mRAMP2/mCLR-expressing cells the EC50 of AM was 0.8 ± 0.2 nM. However, in cells expressing mRAMP2/mCLR-(Δ14-20) up to 10-6 M AM failed to stimulate cAMP production. In mRAMP2/mCLR-(14-20A) expressing cells the cAMP response to AM was minimally restored, and the EC50 was >100 nM. In conclusion, the deletion of the amino acid sequence TRNKIMT of the extreme N terminus of the mCLR maintained CGRP receptor function of mRAMP1/receptor heterodimers, but AM no longer activated the mutant mCLR-(Δ14-20) in the presence of mRAMP2. The TRNKIMT sequence is required for normal mCLR/mRAMIP2 association, and as a consequence, high affinity AM binding signaling the activation of adenylyl cyclase.