A critical role for the short intracellular C terminus in receptor activity-modifying protein function.
Receptor activity-modifying proteins (RAMPs) interact with and modify the behavior of the calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR). We have examined the contribution of the short intracellular C terminus, using constructs that delete the last eight amino acids of each RAMP. C-Terminal deletion of individual RAMPs had little effect on the signaling profile induced when complexed with CLR in COS-7 or human embryonic kidney (HEK)293 cells. Likewise, confocal microscopy revealed each of the mutant RAMPs translocated hemagglutinin-tagged CLR to the cell surface. In contrast, a pronounced effect of RAMP C-terminal truncation was seen for RAMP/CTRa complexes, studied in COS-7 cells, with significant attenuation of amylin receptor phenotype induction that was stronger for RAMP1 and -2 than RAMP3. The loss of amylin binding upon C-terminal deletion could be partially recovered with overexpression of Galpha(s), suggesting an impact of the RAMP C terminus on coupling of G proteins to the receptor complex. In HEK293 cells the c-Myc-RAMP1 C-terminal deletion mutant showed high receptor-independent cell surface expression; however, this construct showed low cell surface expression when expressed alone in COS-7 cells, indicating interaction of RAMPs with other cellular components via the C terminus. This mutant also had reduced cell surface expression when coexpressed with CTR. Thus, this study reveals important functionality of the RAMP C-terminal domain and identifies key differences in the role of the RAMP C terminus for CTR versus CLR-based receptors.
Udawela
Madhara
M
Drug Discovery Biology Laboratory, Department of Pharmacology, Bldg. 13E, Monash University, Clayton, 3800 Victoria, Australia.
Christopoulos
George
G
Morfis
Maria
M
Christopoulos
Arthur
A
Ye
Siying
S
Tilakaratne
Nanda
N
Sexton
Patrick M
PM
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism; D000939:Epitopes / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism*; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D010641:Phenotype; D011485:Protein Binding; D051381:Rats; D058262:Receptor Activity-Modifying Protein 1; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D058265:Receptors, Adrenomedullin; D018003:Receptors, Calcitonin / Q000378:metabolism; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000378:metabolism; D058287:Receptors, Islet Amyloid Polypeptide; D018000:Receptors, Peptide / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014162:Transfection
D000939:Epitopes; D047908:Intracellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D050505:Mutant Proteins; C545502:RAMP1 protein, human; C545504:RAMP3 protein, human; D058262:Receptor Activity-Modifying Protein 1; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D058265:Receptors, Adrenomedullin; D018003:Receptors, Calcitonin; D018015:Receptors, Calcitonin Gene-Related Peptide; D058287:Receptors, Islet Amyloid Polypeptide; D018000:Receptors, Peptide; D000242:Cyclic AMP; D019205:GTP-Binding Protein alpha Subunits, Gs