The transmembrane domain of receptor-activity-modifying protein 1 is essential for the functional expression of a calcitonin gene-related peptide receptor.
Three receptor-activity-modifying proteins (RAMP) define specific interactions between calcitonin (CT) gene-related peptide (CGRP), adrenomedullin (AM) and amylin, and a CT receptor or a CT receptor-like receptor (CRLR). Both form heterodimeric RAMP/receptor complexes at the cell surface. This association represents a novel principle of G protein-coupled receptor function. RAMP1 is transported to the cell surface together with the CRLR or the CT receptor. Here, we have investigated the functional relevance of the short C-terminal intracellular tail QSKRTEGIV and of the single transmembrane domain of human (h) RAMP1 for their interactions with the hCRLR to constitute a CGRP receptor. To this end, hRAMP1 has been sequentially truncated from the C-terminus, and [(125)I]h alpha CGRP/hRAMP1/hCRLR association at the cell surface and cAMP accumulation in response to h alpha CGRP have been examined. With the C-terminal truncation of hRAMP1 by four amino acids wild-type hRAMP1 function was maintained, and the hCRLR was required for the transport of hRAMP1 to the cell surface. Further truncation of hRAMP1 through removal of the remaining five intracellular amino acids revealed CRLR-independent cell surface delivery but otherwise normal hRAMP1 activity. Sequential shortening of the hRAMP1 transmembrane domain resulted in progressively impaired association with the hCRLR and, as a consequence, abolished CGRP receptor function. In conclusion, the intracellular QSKRT sequence adjacent to the transmembrane domain of hRAMP1 provides a signal for intracellular retention. The sequence is unrelated to consensus endoplasmic reticulum retention/retrieval motives and overridden by the presence of the hCRLR. The entire single transmembrane domain of hRAMP1 together with one hydrophilic amino acid residue at its C-terminus is required for the formation of a fully functional CGRP/hRAMP1/hCRLR receptor complex.
Steiner
Sarah
S
Research Laboratory for Calcium Metabolism, Department of Orthopedic Surgery, University of Zurich, Klinik Balgrist, 8008 Zurich, Switzerland.
Muff
Roman
R
Gujer
Remo
R
Fischer
Jan A
JA
Born
Walter
W
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D015740:Calcitonin Gene-Related Peptide / Q000494:pharmacology; D002522:Chlorocebus aethiops; D003432:Cross-Linking Reagents; D000242:Cyclic AMP / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010957:Plasmids; D058262:Receptor Activity-Modifying Protein 1; D058261:Receptor Activity-Modifying Proteins; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion
D003432:Cross-Linking Reagents; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; C545502:RAMP1 protein, human; D058262:Receptor Activity-Modifying Protein 1; D058261:Receptor Activity-Modifying Proteins; D018015:Receptors, Calcitonin Gene-Related Peptide; D011994:Recombinant Proteins; D000242:Cyclic AMP; D015740:Calcitonin Gene-Related Peptide