Structure/function studies on the activation of the rainbow trout melanocortin-2 receptor.
Functional expression of the rainbow trout (rt) melanocortin-2 receptor (MC2R) in CHO cells requires co-expression with a teleost melanocortin-2 receptor accessory protein (MRAP) such as zebrafish (zf) MRAP. Transiently transfected rtMC2R/zfMRAP1 CHO cells were used to evaluate the efficacy of alanine substituted analogs of hACTH(1-24) in three motifs in the ligand: H(6)F(7)R(8)W(9), G(10)K(11)P(12)V(13)G(14), and K(15)K(16)R(17)R(18)P(19). Alanine substitution at all positions in each motif either completely blocked activation of the receptor (H(6)F(7)R(8)W(9) and K(15)K(16)R(17)R(18)P(19)) or resulted in just over 400 fold increase in EC50 value (G(10)K(11)P(12)V(13)G(14)). Single alanine substitutions in the H(6)F(7)R(8)W(9) motif indicated that substitution at either W(9) or R(8) resulted in a much larger increase in EC50 values as compared to substitutions at either F(7) or W(9). Alanine substitution at either K(15)K(16) or R(17)R(18)P(19) in the K(15)K(16)R(17)R(18)P(19) motif resulted in a statistically equivalent increase in EC50 value of at least 600 fold. Finally, alanine substitutions in the G(10)K(11)P(12)V(13)G(14) motif resulted in increases in EC50 values presumably as a result of altering the secondary structure of the ligand. However, truncated analogs of hACTH(1-24) in which either G(10)G(14) (ACTH(1-22), or K(11)P(12)V(13) (ACTH(1-21) were removed had no stimulatory activity. Finally, some of the hACTH(1-24) analogs were tested using rainbow trout head kidney pieces in vitro to confirm whether the response to analogs seen with the transient transfected rtMC2R CHO cells was similar to that of trout interrenal cells. The results of these alanine substitution analog studies are used to construct a multistep hypothetical model for the activation of teleost and tetrapod MC2Rs to account for the unique ligand selectivity of this receptor.
University of Denver, Department of Biological Sciences, Denver, CO 80210, USA.
Liang
L
Liang
University of Denver, Department of Biological Sciences, Denver, CO 80210, USA.
Kristin
K
Schmid
University of Calgary, Department of Biological Sciences, Calgary, Canada.
Navdeep
N
Sandhu
University of Denver, Department of Biological Sciences, Denver, CO 80210, USA.
Joseph K
JK
Angleson
University of Calgary, Department of Biological Sciences, Calgary, Canada.
Mathilakath M
MM
Vijayan
University of Denver, Department of Biological Sciences, Denver, CO 80210, USA. Electronic address: rdores@du.edu.
Robert M
RM
Dores
D000324:Adrenocorticotropic Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D060226:Head Kidney; D008024:Ligands; D017686:Oncorhynchus mykiss / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D044103:Receptor, Melanocortin, Type 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D008024:Ligands; D044103:Receptor, Melanocortin, Type 2; D000324:Adrenocorticotropic Hormone
ACTH; Melanocortin-2 receptor; Rainbow trout