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Enhanced Activity in Parathyroid Hormone-(1–14) and -(1–11): Novel Peptides for Probing Ligand-Receptor Interactions¹

Endocrine Unit (M.S., P.H.C., A.K., J.T.P., T.J.G.) and Biopolymer Core Facility (A.K.), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Dr. Thomas J. Gardella, Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114.

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)NH₂ 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)NH₂ can be enhanced by using nonnatural amino acids that increase the length and polarizability of the position 11 side-chain. Thus, in LLC-PK₁ cells stably expressing high levels of the human P1Rc, [homoarginine([Har)¹¹]PTH-(1–14)NH₂ was 30-fold more potent for cAMP production than was native PTH-(1–14)NH₂. Combining the homoarginine-11 substitution with other recently identified activity-enhancing substitutions yielded [Ala^3,12,Gln¹⁰,Har¹¹,Trp¹⁴]PTH-(1–14)NH₂, which was 1500-fold more potent than PTH-(1–14)NH₂ (EC₅₀ = 0.12 ± 0.04 and 190 ± 20 µM, respectively) and only 63-fold less potent than PTH-(1–34) (EC₅₀ = 1.9 ± 0.5 nM). The even shorter analog [Ala³,Gln¹⁰,Har¹¹]PTH-(1–11)NH₂ was also a full cAMP agonist (EC₅₀ = 3.1 ± 1.5 µM). Receptor mutations at Phe¹⁸⁴ and Leu¹⁸⁷ 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.

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