Enhanced activity in parathyroid hormone-(1-14) and -(1-11): novel peptides for probing ligand-receptor interactions.
The amino-terminal portion of PTH is critical for PTH-1 receptor (P1Rc) activation. In exploring this component of the ligand receptor interaction, we recently showed that the agonist potency of the weakly active PTH-(1-14)NH(2) peptide can be enhanced by natural amino acid substitutions at several positions, including position 11 (normally leucine). Here we show that the potency of PTH-(1-14)NH(2) can be enhanced by using nonnatural amino acids that increase the length and polarizability of the position 11 side-chain. Thus, in LLC-PK(1) cells stably expressing high levels of the human P1Rc, [homoarginine([Har)(11)]PTH-(1-14)NH(2) was 30-fold more potent for cAMP production than was native PTH-(1-14)NH(2). Combining the homoarginine-11 substitution with other recently identified activity-enhancing substitutions yielded [Ala(3,12),Gln(10),Har(11),Trp(14)]PTH-(1-14)NH(2), which was 1500-fold more potent than PTH-(1-14)NH(2) (EC(50) = 0.12 +/- 0.04 and 190 +/- 20 microM, respectively) and only 63-fold less potent than PTH-(1-34) (EC(50) = 1.9 +/- 0.5 nM). The even shorter analog [Ala(3),Gln(10),Har(11)]PTH-(1-11)NH(2) was also a full cAMP agonist (EC(50) = 3.1 +/- 1.5 microM). Receptor mutations at Phe(184) and Leu(187) located near the boundary of the amino-terminal domain and transmembrane domain-1 severely impaired responsiveness to the PTH-(1-11) analog. Overall, these studies demonstrate that PTH analogs of only 11 amino acids are sufficient for activation of the PTH-1 receptor through interaction with its juxtamembrane region.
Shimizu
M
M
Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
Carter
P H
PH
Khatri
A
A
Potts
J T
JT
Gardella
T J
TJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D000242:Cyclic AMP / Q000096:biosynthesis; D006801:Humans; D018374:LLC-PK1 Cells; D008024:Ligands; D010281:Parathyroid Hormone / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D018016:Receptors, Parathyroid Hormone / Q000187:drug effects* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013552:Swine; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism
D008024:Ligands; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D018016:Receptors, Parathyroid Hormone; C430894:parathyroid hormone (1-11)amide, Ala(3)-Gln(10)-Har(11)-; C430893:parathyroid hormone (1-14)amide; D000242:Cyclic AMP; D010738:Type C Phospholipases