Molecular and Pharmacological Characterization of the Rat RAMP2B Splice Variant
Receptor activity-modifying protein 2 (RAMP2) forms a hetero-oligomer with the calcitonin receptor-like receptor (CL) homo-oligomer to produce the adrenomedullin receptor 1 (AM1). RAMP2 has also been shown to associate with other receptors including the calcitonin receptor (CTR), vasoactive intestinal peptide/pituitary adenylate cyclase activating peptide (VPAC) receptor 1, parathyroid hormone (PTH) receptor 1 and glucagon receptor. An adrenomedullin receptor (AM2) is also formed by the RAMP3 and CL complex. A partial sequence of a rat RAMP2 splice variant was previously identified in our laboratory. I hypothesized that human tissue expresses RAMP2 splice variants and that a clone of the full-length rat RAMP2 splice variant forms an adrenomedullin receptor with decreased function in the presence of CL. I used RT-PCR to identify a human RAMP2 splice variant containing an additional fifteen nucleotides and five putative amino acid residues in the N-terminus of the accessory protein. The region of the splice variant does not have a specific, known purpose but is located in the variable N-terminus region. The full-length rat RAMP2b was cloned using RT-PCR and has a twenty-six amino acid deletion in a highly hydrophobic region of the signal peptide. SignalP version 3.0 and Kyte-Doolittle hydropathy plots predict a low probability that the RAMP2b has a signal peptide. Confocal microscopy was used to determine whether the rat RAMP2b trafficked to the plasma membrane. I transiently co-transfected COS-7 cells with an N-terminal HA-tagged CL and either the wild-type RAMP2, containing the intact signal peptide, or the RAMP2b. Non-permeabilized live cells were immunostained with HA and RAMP2 primary antibodies to determine if epitopes of the proteins were present on the outer surface of the plasma membrane. My studies showed that both the rat RAMP2 and RAMP2b trafficked to the cell membrane in the presence or absence of CL. After determining that the rRAMP2b did traffic to the cell membrane, the adrenomedullin receptor function was studied by stimulating the co-transfected COS-7 cells with adrenomedullin and measuring the cAMP levels. The rRAMP2b and hCL transfected COS-7 cells showed a decrease in adrenomedullin receptor activity.
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